Reduction of porcine circovirus-2 viral load with inactivated PCV-2

ABSTRACT

Porcine circovirus-2 (PCV-2) is a recently identified agent wherein the potential spectrum of PCV-2-associated disease has been expanded by evidence of vertical and sexual transmission and associated reproductive failure in swine populations. PCV-2 was isolated from a litter of aborted piglets from a farm experiencing late term abortions and stillbirths. Severe, diffuse myocarditis was present in one piglet associated with extensive immunohistochemical staining for PCV-2 antigen. Variable amounts of PCV-2 antigen were also present in liver, lung and kidney of multiple fetuses. Inoculation of female pigs with a composition including an immunogen from PCV-2 or an epitope of interest from such an immunogen or with a vector expressing such an immunogen or epitope of interest prior to breeding, such as within the first five weeks of life, or prior to the perinatal period, or repeatedly over a lifetime, or during pregnancy, such as between the 6 th  and 8 th  and/or the 10 th  and 13 th  weeks of gestation, can prevent myocarditis, abortion and intrauterine infection associated with porcine circovirus-2. In addition, innoculation of male and/or female pigs with the aforementioned compositions can be carried out to prevent transmission of PCV-2 from male to female (or vice versa) during mating. Thus, the invention involves methods and compositions for preventing myocarditis, abortion and intrauterine infection associated with porcine circovirus-2.

RELATED APPLICATIONS

This application claims priority from and is based upon U.S applicationSer. No. 60/151,564, filed Aug. 31, 1999. Reference is made to:WO-A-01409, published from PCT/EP99/04698, filed Jun. 28, 1999 and U.S.application Ser. No. 09/347,594, filed Jul. 1, 1999, both claimingpriority from French application No. 98 08777, filed Jul. 6, 1998; U.S.application Ser. No. 09/161,092, filed Sep. 25, 1998 as acontinuation-in-part of U.S. application Ser. No. 09/082,558, filed May21, 1998, claiming priority from French applications Nos. 97 12382, 9800873 and 98 03707, filed Oct. 3, 1997, Jan. 22, 1998 and Mar. 20, 1998,respectively; WO-A-99 18214; and the U.S. applications of Audonnet etal. and Bublot et al., Ser. Nos. 60/138,352 and 60/138,478,respectively, both filed Jun. 10, 1999 (“DNA VACCINE-PCV”, and “PORCINECIRCOVIRUS RECOMBINANT POXVIRUS VACCINE”). Reference is additionallymade to each of the documents cited in the text and in the record orprosecution of each of the aforementioned U.S. and French applications,including without limitation WO 98/03658, published Jan. 29, 1998 fromPCT/FR97/01313, filed Jul. 15, 1997 and designating the U.S. andclaiming priority from French application 96 09338, filed Jul. 19, 1996(the U.S. continuation-in-part of PCT/FR97/01313 being U.S. applicationSer. No. 09/232,468, filed Jan. 15, 1999 for “POLYNUCLEOTIDE VACCINEFORMULA AGAINST PORCINE REPRODUCTIVE AND RESPIRATORY PATHOLOGIES”).Mention is also made of PCT WO99/29717. Each of the aforementioned U.S.,PCT and French applications (including parenthetically), and eachdocument cited in the text and the record or prosecution of each of theaforementioned U.S., PCT and French applications (includingparenthetically) (“application cited documents”) and each document citedor referenced in each of the application cited documents, is herebyincorporated herein by reference; and, technology in each of theaforementioned U.S., PCT and French applications (includingparenthetically), and each document cited in the text and the record orprosecution of each of the aforementioned U.S., PCT and Frenchapplications (including parenthetically) can be used in the practice ofthis invention.

FIELD OF THE INVENTION

The invention relates to methods and/or compositions for the preventionand/or treatment of PCV-2-caused myocarditis, and/or abortion and/orintrauterine infection, as well as pathologic sequelae including but notlimited to post-weaning multisystemic wasting syndrome; and, to methodsfor preparing such compositions and kits for preparing such compositionsor for performing such methods, inter alia.

Various documents are cited in this text. Citations in the text can beby way of a citation to a document in the reference list, e.g., by wayof an author(s) and document year citation to a document listed in thereference list, or by full citation in the text to a document that mayor may not also be listed in the reference list.

There is no admission that any of the various documents cited in thistext are prior art as to the present invention. Any document having asan author or inventor person or persons named as an inventor herein is adocument that is not by another as to the inventive entity herein. Alldocuments cited in this text (“herein cited documents”) and alldocuments cited or referenced in herein cited documents are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Porcine circovirus-2 (PCV-2) was recently identified as an agent thathas been consistently associated with post-weaning multisystemic wastingsyndrome (PMWS) in swine populations in several parts of the world(Allan et al. 1998; Ellis et al., 1998). Isolates of PCV-2 obtained frominfected pigs in several countries are virtually identical genetically,and are distinctly different from the PCV (CCL33, PCV-1) that wasoriginally identified in the 1970's as a noncytopathic contaminant ofporcine kidney (PK/15) cell line (Meehan et al. 1998; Tischer et al.1974). Pigs with naturally acquired or experimentally induced PCV-2infections present with progressive weight loss, tachypnea, dyspnea, andjaundice (Allan et al. 1998; Allan et al. 1999; Ellis et al. 1998; Elliset al. 1999). Gross pathologic findings that have been directlyassociated with PCV-2 antigen include, lymphadenopathy, interstitialpneumonia, hepatitis and nephritis (Allan et al. 1998; Allan et al.1999; Ellis et al. 1998; Ellis et al. 1999). PCV-2 has not heretoforebeen directly linked to abortion or lesions in fetal pigs. Thus,heretofore, it has not been proposed to address the issue ofPCV-2-caused myocarditis, and/or abortion and/or intrauterine infection.

OBJECTS AND SUMMARY OF THE INVENTION

It has surprisingly been found that PCV-2 is a causative agent ofmyocarditis, abortion and intrauterine infection, as well aspost-weaning multisystemic wasting syndrome.

By definition, a PCV-2 immunogen is intended to encompass liveattenuated or inactivated PCV-2, or subunit(s) from PCV-2 obtained by invitro expression or by extraction, or fragment(s) comprising at leastone epitope of interest which can be obtained by chemical synthesis orby in vitro recombinant expression, as well as recombinant vector(s)comprising and expressing in vivo sequence(s) or fragment(s) orepitope(s) of PCV-2 genome as herein disclosed or as in documents citedor referenced herein.

A similar definition applies for an immunogen of another porcinepathogen as disclosed herein.

Thus, an object of the invention can be to provide methods and/orcompositions for the prevention and/or treatment of PCV-2-causedmyocarditis, and/or abortion and/or intrauterine infection, as well aspost-weaning multisystemic wasting syndrome and/or pathologic sequelaeincluding but not limited to post-weaning multisystemic wastingsyndrome; and, methods for formulating such compositions and uses of aPCV-2 immunogen (which compositions can also include a porcineparvovirus (PPV) immunogen, wherein when recombinant vector expressionis used, the vector can co-express both the PPV and the PCV-2immunogens, inter alia) for formulating such compositions.

Another object of the invention is the isolation and characterisation ofnew PCV-2 strains identified 1103 (1103/1 P.2) and 1121 (1121/1 P.1),and their uses to produce immunogens, as well as antigens and antibodiesfor diagnostics, in relation with PCV-2-caused myocarditis, and/orabortion and/or intrauterine infection, as well as post-weaningmultisystemic wasting syndrome and/or pathologic sequelae associatedtherewith.

The invention provides also for inoculation of female pigs (e.g., sows,gilts) with a composition comprising a (at least one) PCV-2 immunogen(which composition can also include an immunogen from porcineparvovirus) prior to breeding; and/or prior to serving, and/or duringgestation (or pregnancy); and/or prior to the perinatal period orfarrowing; and/or repeatedly over a lifetime , to prevent myocarditisand/or abortion and/or intrauterine infection associated with PCV-2, aswell as post-weaning multisystemic wasting syndrome and other pathologicsequelae associated with PCV-2; or, to elicit an immunogenic orprotective response against PCV-2 and thereby prevent post-weaningmultisystemic wasting syndrome and/or myocarditis and/or abortion and/orintrauterine infection associated with porcine circovirus-2 and/or otherpathologic sequelae associated with PCV-2.

Advantageously, at least one inoculation is done before serving. It isalso advantageously followed by an inoculation to be performed duringgestation, e.g., at about mid-gestation (at about 6-8 weeks ofgestation) and/or at the end of gestation (at about 11-13 weeks ofgestation). Thus, an advantageous regimen is an inoculation beforeserving and a booster inoculation during gestation. Thereafter, therecan be reinoculation before each serving and/or during gestation atabout mid-gestation (at about 6-8 weeks of gestation) and/or at the endof gestation (at about 11-13 weeks of gestation). Preferably,reinoculation can be during gestation only.

In another preferred embodiment, piglets, such as piglets fromvaccinated females (e.g., inoculated as herein discussed), areinoculated within the first weeks of life, e.g., inoculation at oneand/or two and/or three and/or four and/or five weeks of life. Morepreferably, piglets are first inoculated within the first week of lifeor within the third week of life (e.g., at the time of weaning). Evenmore advantageous, such piglets are then boosted two (2) to four (4)weeks later (after being first inoculated). Thus, both offspring, aswell as female pig (e.g., sow, gilt) can be administered compositions ofthe invention and/or can be the subject of performance of methods of theinvention.

Thus, the invention also comprehends immunogenic or vaccine compositionsfor preventing or treating myocarditis and/or abortion and/orintrauterine infection associated with porcine circovirus-2, as well aspost-weaning multisystemic wasting syndrome and other pathologicsequelae associated with PCV-2. An immunogenic (or immunological)composition elicits an immunological response—local or systemic. Avaccine composition elicits a local or systemic protective response. Theterms “immunological composition” and “immunogenic composition” includea “vaccine composition” (as the two former terms can be protectivecompositions). The composition can comprise a PCV-2 immunogen (whichcomposition can. also include a PPV immunogen).

And, the invention further comprehends uses of a PCV-2 immunogen (whichcomposition can also include a PPV immunogen) to formulate animmunogenic or vaccine composition for preventing or treatingmyocarditis and/or abortion and/or intrauterine infection associatedwith porcine circovirus-2, as well as post-weaning multisystemic wastingsyndrome and other pathologic sequelae associated with PCV-2.

Further still, the invention comprehends an immunogenic or vaccinecomposition for the prevention and/or treatment of PCV-2-causedmyocarditis, and/or abortion and/or intrauterine infection and/orpost-weaning multisystemic wasting syndrome comprising apharmaceutically or veterinarily acceptable carrier and/or vehicleand/or excipient and/or adjuvant, and a PCV-2 immunogen

The composition can additionally include at least one immunogen from atleast one additional pig pathogen, e.g.: Porcine Reproductive andRespiratory Syndrome (PRRS), Mycoplasma hyopneumoniae, Actinobacilluspleuropneumoniae, E. coli, Bordetella bronchiseptica, Pasteurellamultocida, Erysipelothrix rhusiopathiae, Pseudorabies, Hog cholera,Swine Influenza, and Porcine Parvovirus (PPV). Thus, vector-basedcompositions can include at least one immunogen from at least oneadditional pig pathogen, such as a vector expressing a sequence fromthis pathogen, wherein the vector can also be the vector expressing thePCV-2 immunogen. The vector expressing a PCV-2 sequence can comprise aPCV-2 sequence or fragment thereof as herein disclosed or as indocuments cited or referenced herein; and the invention comprehends suchnucleic acid molecules, vectors containing them, compositions comprisingsuch nucleic acid molecules or vector expression products from suchnucleic acid molecules, compositions comprising such expressionproducts, probes or primers for such nucleic acid molecules, and methodsfor making and using any or all of the foregoing.

The vector can comprise a DNA vector plasmid, a bacteria such as an E.coli, a virus such as baculovirus, a herpesvirus including pig herpesviruses, including Aujeszky's disease virus, an adenovirus including aporcine adenovirus, a poxvirus, including a vaccinia virus, an avipoxvirus, a canarypox virus, a racoonpox and a swinepox virus, and thelike. The vector-based compositions can comprise a vector that containsand expresses an ORF selected from the group consisting of ORFs 1 to 13,such as an ORF selected from ORFs 4, 7, 10 and 13; preferably ORFs 4and/or 13, of a PCV-2, advantageously of any one of the PCV-2 strainsidentified herein. And, the immunogen in compositions (either PCV-2and/or from another pig pathogen) can be recombinantly produced. Theword plasmid is intended to include any DNA transcription unit in theform of a polynucleotide sequence comprising the PCV sequence to beexpressed. Advantageously, the plasmid includes elements necessary forits expression; for instance, expression in vivo. The circular plasmidform, supercoiled or otherwise, is advantageous; and, the linear form isalso included within the scope of the invention. The plasmid immunogenicor vaccine composition can be administered by way of a gene gun,intradermally via an needleless injector, subcutaneously orintramuscularly, or by mucosal route, or by any other means that allowsfor expression in vivo, and advantageously an immunogenic or protectiveresponse.

It is noted that the expression product generated by vectors orrecombinants in this invention optionally can also be isolated and/orpurified from infected or transfected cells; for instance, to preparecompositions for administration to pigs; however, in certain instances,it may be advantageous not to isolate and/or purify an expressionproduct from a cell; for instance, when the cell or portions thereofenhance the immunogenic effect of the polypeptide. And, techniques forprotein purification and/or isolation from this disclosure and documentscited herein, inter alia, and thus within the ambit of the skilledartisan, can be used, without undue experimentation, to purify and/orisolate recombinant or vector expression products and/or subunits ofPCV-2 and/or other pig pathogens, in the practice of the invention, andsuch techniques, in general, can include: precipitation by takingadvantage of the solubility of the protein of interest at varying saltconcentrations, precipitation with organic solvents, polymers and othermaterials, affinity precipitation and selective denaturation; columnchromatography, including high performance liquid chromatography (HPLC),ion-exchange, affinity, immunoaffinity or dye-ligand chromatography;immunoprecipitation, gel filtration, electrophoretic methods,ultrafiltration and isoelectric focusing, and their combinations, interalia.

The invention further envisages methods for the prevention and/ortreatment of porcine circovirus-2 (PCV-2)-caused myocarditis, and/orabortion and/or intrauterine infection and/or post-weaning multisystemicwasting syndrome and/or other pathologic sequelae associated with PCV-2comprising inducing an immunogenic or protective response against PCV-2in a pig comprising administering to the pig an aforementioned or hereindisclosed composition.

Thus, the invention comprehends a method for the prevention and/ortreatment of porcine circovirus-2 (PCV-2)-caused myocarditis, and/orabortion and/or intrauterine infection and/or post-weaning multisystemicwasting syndrome and/or other pathologic sequelae associated with PCV-2comprising inducing an immunogenic or protective response against PCV-2in a pig comprising administering to the pig a composition comprising apharmaceutically or veterinarily acceptable carrier or excipient orvehicle, with preferably an adjuvant, and an active agent comprising aPCV-2 immunogen. The method can be for the prevention of PCV-2-causedmycarditis and/or abortion and/or intrauterine infection comprisingadministering a composition comprising a pharmaceutically orveterinarily acceptable carrier and a PCV-2 immunogen. The PCV-2immunogen can be an attenuated live whole PCV-2 or inactivated PCV-2.The method can involve a composition that is a subunit immunogenic, orvaccine composition. The method can involve the composition additionallyincluding at least one immunogen from at least one additional pigpathogen , including a vector expressing such an immunogen or epitope;e.g., the at least one additional pig pathogen can be selected from thegroup consisting of PRRS, Mycoplasma hyopneumoniae, Actinobacilluspleuropneumoniae, E. coli, Pseudorabies, Hog cholera, Bordetellabronchiseptica, Pasteurella multocida, Erysipelothrix rhusiopathiae,Swine Influenza, and PPV and combinations thereof. The method caninvolve a vector that is a DNA vector plasmid, a bacteria such as an E.coli, a virus such as baculovirus, a herpesvirus including Aujeszky'sdisease virus, an adenovirus including a porcine adenovirus, a poxvirus,including a vaccinia virus, an avipox virus, a canarypox virus, and aswinepox virus, and the like. The method can involve a vector-basedcomposition additionally including at least one sequence, fragment orepitope from at least one additional pig pathogen, or a vectorexpressing such a sequence, fragment or epitope, wherein the vector canalso be the vector expressing the PCV-2 sequence, fragment or epitope.The method can involve a vector that contains and expresses an ORFselected from the group consisting of ORFs 1 to 13, e.g., an ORFselectred from ORFs 4, 7, 10 and 13; preferably ORFs 4 and/or 13. Themethod can also involve an immunogen based composition wherein one ormore of the immunogen(s) is recombinantly produced. In this method,females and/or piglets are inoculated as described above.

In another embodiment, the invention involves a method for preparing anyof the aforementioned or herein disclosed compositions comprisingadmixing the pharmaceutically or veterinarily acceptable carrier and thePCV-2 immunogen. The method can further include transfecting orinfecting a cell or host with a recombinant vector that contains DNAencoding a PCV-2 immunogen and expresses that immunogen; and optionallypurifying and/or isolating the immunogen from the cell. Similarly themethod can include isolating and/or purifying a PCV-2 immunogen fromPCV-2, or isolating PCV-2 from a sample.

The invention also provides a kit for preparing any of theaforementioned or herein disclosed compositions or for performing any ofthe aforementioned or herein disclosed methods comprising in a firstcontainer the pharmaceutically or veterinarily acceptable carrier orvehicle or excipient and in a second container the active agentcomprising the PCV-2 immunogen, wherein the first and second containersare optionally packaged together, and the kit optionally includesinstructions for admixture of ingredients of the composition and/oradministration of the composition.

In yet another embodiment, the invention provides for administering anyof the aforementioned or herein disclosed compositions to male and/orfemale pigs; to prevent transmission of PCV-2 and prevent or treat orcontrol myocarditis and/or abortion and/or intrauterine infectionassociated with porcine circovirus-2, as well as post-weaningmultisystemic wasting syndrome and other pathologic sequelae associatedwith PCV-2. Administration is preferably done as described above.

The term “comprising” in this disclosure can mean “including” or canhave the meaning commonly given to the term “comprising” in U.S. PatentLaw.

Other aspects of the invention are described in or are obvious from (andwithin the ambit of the invention) the following disclosure.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts SEQ ID No. 1, the PCV DNA sequence of the genome of theImp. 1011-48121 strain.

FIG. 2 depicts SEQ ID No. 2, the DNA sequence of the genome of the Imp.1011-48285 strain.

FIG. 3 depicts SEQ ID No. 3, the DNA sequence of the genome of the Imp.999 strain.

FIG. 4 depicts SEQ ID No. 4, the DNA sequence of the genome of the Imp.1010 strain.

FIG. 5 depicts SEQ ID No. 5, the DNA sequence of the genome of the PK/15strain.

FIG. 6 depicts SEQ ID No. 6, the DNA sequence of the genome of the Imp.999 strain as defined in the first filing in France on Oct. 3, 1997.

FIG. 7 depicts SEQ ID No. 7, the DNA sequence of the genome of the 1103strain, isolated in Alberta, Canada. k means g (G) or t (T), y means c(C) or t (T). These variations of sequence were observed in the viralpopulation.

FIG. 8 depicts SEQ ID No. 8, the DNA sequence of the genome of the 1121strain, isolated in Saskatoon, Canada.

DETAILED DESCRIPTION

Porcine circovirus-2 (PCV-2) is an agent associated with post-weaningmultisystemic wasting syndrome (PMWS) in swine populations. As shown inExamples 1 and 2, the potential spectrum of disease associated withPCV-2 is expanded by evidence of vertical transmission and associatedreproductive failure.

In particular, Example 1 shows that PCV-2 was isolated from a litter ofaborted piglets from a farm experiencing late term abortions andstillbirths. Severe, diffuse myocarditis was present in one pigletassociated with extensive immunohistochemical staining for PCV-2antigen. Variable amounts of PCV-2 antigen were also present in liver,lung and kidney of multiple fetuses. The presence of other agents thathave been associated with fetal lesions and abortion in swine includingporcine parvovirus, porcine reproductive respiratory syndrome virus,encephalomyocarditis virus and enterovirus could not be established.

More in particular, Example 2 shows that tissues obtained from 30 highhealth herds over a four-year period, and tested in routine cases ofabortion or reproductive failure, were positive for PCV-2 in twosubmissions involving several stillborn piglets and non-viable neonatespresenting with severe diffuse myocarditis, cardiac hypertrophy andevidence of chronic passive congestion. The two positive submissionswere from the same farm, but occurred at two different times. Thepresence of PCV-2 in the hearts and other tissues of affected pigletswas confirmed by immunohistochemistry and virus isolation. Failure todetect porcine circoviruses in cases of reproductive failure prior to1999 in areas of endemic infections supports the view that reproductivedisease is a new clinical manifestation of PCV-2 infection, and furthersuggests that sexual, as well as vertical, modes of transmission areresponsible for viral dissemination in the pig population.

Accordingly, inoculation of pigs, e.g., female pigs, such as sows orgilts, with a composition including at least one PCV-2 immunogen (e.g.from at least one strain chosen among strains Imp 1008, Imp 1010, Imp999, Imp 1011-48285, Imp 1011-48121, 1103 and 1121) (which compositioncan also include at least one immunogen from at least one other porcinepathogen such as at least one porcine parvovirus, wherein when a vectoris used the vector can co-express both the PCV-2 immunogen(s) and the atleast one immunogen of the at least one other porcine pathogen, e.g.,PPV immunogen(s), inter alia), in a schedule of immunization asdescribed above, can prevent myocarditis and/or abortion and/orintrauterine infection associated with PCV-2, as well as post-weaningmultisystemic wasting syndrome and other pathologic sequelae associatedwith PCV-2.

Thus, the invention involves methods and compositions using PCV-2immunogen for preventing myocarditis and/or abortion and/or intrauterineinfection associated with porcine circovirus-2, as well as post-weaningmultisystemic wasting syndrome and other pathologic sequelae associatedwith PCV-2. In particular, immunogen from strain 1103 and/or strain 1121is useful for methods and compositions using PCV-2 immunogen forpreventing myocarditis and/or abortion and/or intrauterine infectionassociated with porcine circovirus-2

The PCV-2 immunogen can be any PCV-2 immunogen including anyPCV-2-expressing vector identified in any herein cited document (or anydocument cited in herein cited documents) including any or all of: U.S.application Ser. No. 09/347,594, filed Jul. 1, 1999; French applicationNo. 98 08777, filed Jul. 6, 1998; U.S. application Ser. No. 09/161,092,filed Sep. 25, 1998; U.S. application Ser. No. 09/082,558, filed May 21,1998; French applications Nos. 97 12382, 98 00873 and 98 03707, filedOct. 3, 1997, Jan. 22, 1998 and Mar. 20, 1998, respectively;.WO-A-9918214; the U.S. applications of Audonnet et al. and Bublot et al., Ser.Nos. 60/138,352 and 60/138,478, respectively, both filed Jun. 10, 1999(“DNA VACCINE-PCV”, and “PORCINE CIRCOVIRUS RECOMBINANT POXVIRUSVACCINE”, respectively); and WO99/29717 (all of which and documentscited therein and in the prosecution thereof being hereby incorporatedherein by reference). Thus, the immunogen from PCV-2 including a vectorexpressing such an immunogen can be prepared in accordance with hereincited documents (or documents cited in herein cited documents).

The composition comprising the PCV-2 immunogen employed in the practiceof this invention can be as in any herein cited document (or anydocument cited in herein cited documents) including any or all of: U.S.application Ser. No. 09/347,594, filed Jul. 1, 1999; French applicationNo. 98 08777, filed Jul. 6, 1998; U.S. application Ser. No. 09/161,092,filed Sep. 25, 1998; U.S. application Ser. No. 09/082,558, filed May 21,1998; French applications Nos. 97 12382, 98 00873 and 98 03707, filedOct. 3, 1997, Jan. 22, 1998 and Mar. 20, 1998, respectively; WO-A-9918214; the U.S. applications of Audonnet et al. and Bublot et al., Ser.Nos. 60/138,352 and 60/138,478, respectively, both filed Jun. 10, 1999(“DNA VACCINE-PCV”, and “PORCINE CIRCOVIRUS RECOMBINANT POXVIRUSVACCINE”, respectively); and WO99/29717 (all of which and documentscited therein and in the prosecution thereof being hereby incorporatedherein by reference). Thus, the composition comprising the PCV-2immunogen including the vector expressing PCV-2 immunogen can beprepared as in herein cited documents.

The at least one immunogen from at least one other porcine pathogen canbe as described in any of the aforementioned or herein cited patent orliterature publications (or documents cited therein), or as used inknown porcine vaccines or immunogenic compositions, or as in WO98/03658, published Jan. 29, 1998 from PCT/FR97/01313, filed Jul. 15,1997; or French application 96 09338, filed Jul. 19, 1996; or U.S.application Ser. No. 09/232,468, filed Jan. 15, 1999 for(“POLYNUCLEOTIDE VACCINE FORMULA AGAINST PORCINE REPRODUCTIVE ANDRESPIRATORY PATHOLOGIES”).

The amount of PCV-2 immunogen in compositions employed in the inventioncan be as described in any of the aforementioned or herein cited patentor literature publications (or documents cited therein). And, the amountof at least one immunogen from at least one other porcine pathogen canbe as described in any of the aforementioned or herein patent orliterature publications (or documents cited therein), or as used inknown porcine vaccines or immunogenic compositions.

Compositions for use in the invention can be prepared in accordance withstandard techniques well known to those skilled in the veterinary orpharmaceutical or arts. Such compositions can be administered in dosagesand by techniques well known to those skilled in the veterinary artstaking into consideration such factors as the age, sex. weight,condition and particular treatment of the pig, and the route ofadministration. The compositions can be administered alone, or can beco-administered or sequentially administered with other compositions ofthe invention (e.g., other compositions comprising a PCV-2 immunogen) orwith other prophylactic or therapeutic compositions (e.g., other porcineimmunogenic or vaccine compositions). Thus, the invention also providesmultivalent or “cocktail” or combination compositions and methodsemploying them. In this regard, reference is made to U.S. Pat. No.5,843,456, incorporated herein by reference, and directed to rabiescompositions and combination compositions and uses thereof.

Compositions of the invention may be used for parenteral or mucosaladministration, preferably by intradermal or intramuscular routes. Inparticular for intradermal route, injection can be done using aneedleless injector. When mucosal administration is used, it is possibleto use oral, nasal, or ocular routes.

In such compositions the immunogen(s) may be in a mixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose or the like, and/or preferably with anadjuvant. The compositions can also be lyophilized or frozen. Thecompositions can contain auxiliary substances such as pH bufferingagents, adjuvants, preservatives, polymer excipients used for mucosalroutes, and the like, depending upon the route of administration and thepreparation desired.

Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation. Suitabledosages can also be based upon the text herein and documents citedherein.

Adjuvants are substances that enhance the immune response to immunogens.Adjuvants, can include aluminum hydroxide and aluminum phosphate,saponins e.g., Quil A, water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion. The emulsion can be based in particularon light liquid paraffin oil (European Pharmacopea type); isoprenoid oilsuch as squalane or squalene; oil resulting from the oligomerization ofalkenes, in particular of isobutene or decene; esters of acids or ofalcohols containing a linear alkyl group, more particularly plant oils,ethyl oleate, propylene glycol di(caprylate/caprate), glyceryltri(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. T heemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycerol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic® products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.). JohnWiley and Sons, N.Y., pp51-94 (1995) and Todd et al., Vaccine 15:564-570(1997).

For example, it is possible to use the SPT emulsion described on page147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M.Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book.

For example the adjuvant-containing vaccine is prepared in the followingway: 67% v/v of aqueous phase comprising the immunogen are emulsified in2,3% w/v of anhydromannitol oleate, 2,6% w/v of oleic acid ethoxylatedwith 11 EO (ethylene oxide) and 28,1% v/v of light liquid paraffin oil(European Pharmacopea type) with the aid of an emulsifying turbomixer.

An alternative method for preparing the emulsion consists inemulsifying, by passages through a high-pressure homogenizer, a mixtureof 5% w/v squalane, 2.5% w/v Pluronic® L121, 0.2% w/v of an ester ofoleic acid and of anhydrosorbitol ethoxylated with 20 EO, 92.3% v/v ofthe aqueous phase comprising the immunogen.

It is also possible to formulate with synthetic polymers (e.g., homo-and copolymers of lactic and glycolic acid, which have been used toproduce microspheres that encapsulate immunogens, see Eldridge et al.,Mol. Immunol. 28:287-294 (1993), e.g., biodegradable microspheres), withcytokines such as IL-2 and IL-12 (see, e.g., U.S. Pat. No. 5,334,379),and GMCSF, advantageously porcine GMCSF (granulocyte macrophage-colonystimulating factor; see, generally, U.S. Pat. Nos. 4,999,291 and5,461,663, see also Clark et al., Science 1987, 230:1229; Grant et al.,Drugs, 1992, 53:516), inter alia. Certain adjuvants can be expressed invivo with immunogen(s) and/or epitope(s); e.g., cytokines, GMCSF (see,e.g., Inumaru and Takamatsu, Immunol. Cell. Biol., 1995, 73:474-76concerning a plasmid encoding and expressing porcine GM-CSF).

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked. especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462(incorporated herein by reference) which describes such acrylic polymerscross-linked with a polyhydroxylated compound having at least 3 hydroxylgroups, preferably not more than 8, the hydrogen atoms of at least threehydroxyls being replaced by unsaturated aliphatic radicals having atleast 2 carbon atoms. The preferred radicals are those containing from 2to 4 carbon atoms, e.g. vinyls, allyls and other ethylenicallyunsaturated groups. The unsaturated radicals may themselves containother substituents, such as methyl. The products sold under the nameCarbopol® (BF Goodrich, Ohio, USA) are particularly appropriate. Theyare cross-linked with an allyl sucrose or with allyl pentaerythritol.Among then, there may be mentioned Carbopol® 974P, 934P and 971P. Amongthe copolymers of maleic anhydride and alkenyl derivative, thecopolymers EMA® (Monsanto) which are copolymers of maleic anhydride andethylene, linear or cross-linked, for example cross-linked with divinylether, are preferred. Reference may be made to J. Fields et al., Nature,186: 778-780, 4 June 1960, incorporated herein by reference.

From the point of view of their structure, the polymers of acrylic ormethacrylic acid and the copolymers EMA® are preferably formed of basicunits of the following formula:

in which:

R₁ and R₂, which are identical or different, represent H or CH₃;

x=0 or 1, preferably x=1; and

y=1 or 2, with x+y=2.

For the copolymers EMA®, x=0 and y=2. For the carbomers, x=y=1.

The dissolution of these polymers in water leads to an acid solutionthat will be neutralized, preferably to physiological pH, in order togive the adjuvant solution into which the immunogenic, immunological orvaccine composition itself will be incorporated. The carboxyl groups ofthe polymer are then partly in COO⁻ form.

Preferably, a solution of adjuvant according to the invention,especially of carbomer, is prepared in distilled water, preferably inthe presence of sodium chloride, the solution obtained being at acidicpH. This stock solution is diluted by adding it to the desired quantity(for obtaining the desired final concentration), or a substantial partthereof, of water charged with NaCl, preferably physiological saline(NaCL 9 g/l) all at once in several portions with concomitant orsubsequent neutralization (pH 7.3 to 7.4), preferably with NaOH. Thissolution at physiological pH will be used as it is for mixing with thevaccine, which may be especially stored in freeze-dried, liquid orfrozen form.

The polymer concentration in the final vaccine composition can be 0.01%to 2% w/v, e.g., 0.06 to 1% w/v, such as 0.1 to 0.6% w/v.

From this disclosure and the knowledge in the art, the skilled artisancan select a suitable adjuvant, if desired, and the amount thereof toemploy in an immunological, immunogenic or vaccine composition accordingto the invention, without undue experimentation.

The immunogenic or vaccine compositions according to the invention maybe associated to at least one live attenuated, inactivated, or sub-unitvaccine, or recombinant vaccine (e.g. poxvirus as vector or DNA plasmid)expressing at least one immunogen or epitope of interest from at leastone another pig pathogen.

Compositions in forms for various administration routes are envisionedby the invention. And again, the effective dosage and route ofadministration are determined by known factors, such as age, sex, weightand other screening procedures which are known and do not require undueexperimentation. Dosages of each active agent can be as in herein citeddocuments and/or can range from one or a few to a few hundred orthousand micrograms, e.g., 1 μg to 1 mg, for a subunit immunogenic, orvaccine composition; and, 10⁴ to 10¹⁰ TCID₅₀ advantageously 10⁶ to 10⁸TCID₅₀ for an inactivated (titre before inactivation) immunogenic, orvaccine composition. For a live attenuated immunogenic or vaccinecomposition, the dose can be between 10¹ and 10⁸ TCID₅₀ advantageously10³ and 10⁶ TCID₅₀.

Recombinants or vectors can be administered in a suitable amount toobtain in vivo expression corresponding to the dosages described hereinand/or in herein cited documents. For instance, suitable ranges forviral suspensions can be determined empiracally. The viral vector orrecombinant in the invention can be administered to a pig or infected ortransfected into cells in an amount of about at least 10³ pfu; morepreferably about 10⁴ pfu to about 10¹⁰ pfu, e.g., about 10⁵ pfu to about10⁹ pfu, for instance about 10⁶ pfu to about 10⁸ pfu, per dose, e.g. ofabout 2 ml. And, if more than one gene product is expressed by more thanone recombinant, each recombinant can be administered in these amounts;or, each recombinant can be administered such that there is, incombination, a sum of recombinants comprising these amounts.

In plasmid compositions employed in the invention, dosages can be asdescribed in documents cited herein or as described herein. Forinstance, suitable quantities of each plasmid DNA in plasmidcompositions can be 1 μg to 2 mg, preferably 50 μg to 1 mg. Documentscited herein regarding DNA plasmid vectors may be consulted by theskilled artisan to ascertain other suitable dosages for DNA plasmidvector compositions of the invention, without undue experimentation.

However, the dosage of the composition(s), concentration of componentstherein and timing of administering the composition(s), which elicit asuitable immunologenic response, can be determined by methods such as byantibody titrations of sera, e.g., by ELISA and/or seroneutralizationassay analysis and/or by vaccination challenge evaluation in pig. Suchdeterminations do not require undue experimentation from the knowledgeof the skilled artisan, this disclosure and the documents cited herein.And, the time for sequential administrations can be likewise ascertainedwith methods ascertainable from this disclosure, and the knowledge inthe art, without undue experimentation.

The PCV-2 immunogen can be obtained from PCV-2 or can be obtained fromin vitro recombinant expression of PCV-2 gene(s) or portions or epitopesthereof. Methods for making and/or using vectors (or recombinants) forexpression can be by or analogous to the methods disclosed in: U.S. Pat.Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807,4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235,5,756,103, 5,766,599, 6,004,777, 5,990,091, 6,033,904, 5,869,312,5,382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018,Paoletti, “Applications of pox virus vectors to vaccination: An update,”PNAS USA 93:11349-11353, October 1996, Moss, “Genetically engineeredpoxviruses for recombinant gene expression, vaccination, and safety,”PNAS USA 93:11341-11348, October 1996, Smith et al., U.S. Pat. No.4,745,051 (recombinant baculovirus), Richardson, C. D. (Editor), Methodsin Molecular Biology 39, “Baculovirus Expression Protocols” (1995 HumanaPress Inc.), Smith et al., “Production of Huma Beta Interferon in InsectCells Infected with a Baculovirus Expression Vector,” Molecular andCellular Biology, Dec., 1983, Vol. 3, No. 12, p. 2156-2165; Pennock etal., “Strong and Regulated Expression of Escherichia coliB-Galactosidase in Infect Cells with a Baculovirus vector,” Molecularand Cellular Biology Mar. 1984, Vol. 4, No. 3, p. 399-406; EPA 0 370573, U.S. application Ser. No. 920,197, filed Oct. 16, 1986, EP Patentpublication No. 265785, U.S. Pat. No. 4,769,331 (recombinantherpesvirus), Roizman, “The function of herpes simplex virus genes: Aprimer for genetic engineering of novel vectors,” PNAS USA93:11307-11312, October 1996, Andreansky et al., “The application ofgenetically engineered herpes simplex viruses to the treatment ofexperimental brain tumors,” PNAS USA 93:11313-11318, October 1996,Robertson et al. “Epstein-Barr virus vectors for gene delivery to Blymphocytes,” PNAS USA 93:11334-11340, October 1996, Frolov et al.,“Alphavirus-based expression vectors: Strategies and applications,” PNASUSA 93:11371-11377, October 1996, Kitson et al., J. Virol. 65,3068-3075, 1991; U.S. Pat. Nos. 5,591,439, 5,552,143, WO 98/00166,allowed U.S. applications Ser. Nos. 08/675,556, and 08/675,566 bothfiled Jul. 3, 1996 (recombinant adenovirus), Grunhaus et al., 1992,“Adenovirus as cloning vectors,” Seminars in Virology (Vol. 3) p.237-52, 1993, Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham,Tibtech 8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70, 429-434,PCT WO91/11525, Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561,Science, 259:1745-49, 1993 and McClements et al., “Immunization with DNAvaccines encoding glycoprotein D or glycoprotein B, alone or incombination, induces protective immunity in animal models of herpessimplex virus-2 disease,” PNAS USA 93:11414-11420, October 1996, andU.S. Pat. Nos 5,591,639, 5,589,466, and 5,580,859 relating to DNAexpression vectors, inter alia. See also WO 98/33510; Ju et al.,Diabetologia, 41:736-739, 1998 (lentiviral expression system); Sanfordet al., U.S. Pat. No. 4,945,050; Fischbach et al. (Intracel), WO90/01543; Robinson et al., seminars in IMMUNOLOGY, vol. 9, pp.271-283(1997) (DNA vector systems); Szoka et al., U.S. Pat. No. 4,394,448(method of inserting DNA into living cells); McCormick et al., U.S. Pat.No. 5,677,178 (use of cytopathic viruses); and U.S. Pat. No. 5,928,913(vectors for gene delivery), as well as other documents cited herein. Aviral vector, for instance, selected from pig herpes viruses, such asAujeszky's disease virus, porcine adenovirus, poxviruses, especiallyvaccinia virus, avipox virus, canarypox virus, and swinepox virus, aswell as DNA vectors (DNA plasmids) are advantageously employed in thepractice of the invention.

The expression product from the PCV-2 gene(s) or portions thereof can beuseful for generating antibodies such as monoclonal or polyclonalantibodies that are useful for diagnostic purposes. Similarly,expression product(s) from the PCV-2 gene(s) or portions thereof can beuseful in diagnostic applications.

Further, one skilled in the art can determine an epitope of interest ina PCV-2 immunogen, or in an immunogen of another porcine pathogen,without undue experimentation, from the disclosure herein and theknowledge in the art; see, e.g., WO 98/40500, incorporated herein byreference, regarding general information for determining an epitope ofinterest or an epitopic region of a protein, inter alia.

With particular reference to U.S. application Ser. No. 09/161,092, filedSep. 25, 1998, U.S. application Ser. No. 09/082,558, filed May 21, 1998,French applications Nos. 97 12382, 98 00873 and 98 03707, filed Oct. 3,1997, Jan. 22, 1998 and Mar. 20, 1998, respectively, and, WO-A-99 18214(all incorporated herein by reference), particularly advantageousimmunogenic, immunological or vaccine compositions are: An immunogenicor vaccine composition, collected from a cell culture in vitro which hasbeen infected with a purified preparation of PCV-2, such as a purifiedpreparation of porcine circovirus selected from the group consisting ofthe preparations deposited at the ECACC, under the following references:accession No. V97100219 (strain Imp.1008), No. V97100218 (strainImp.1010) and accession No. V97100217 (strain Imp.999) deposited Oct. 2,1997, accession No. V98011608(strain Imp. 1011-48285) and No. V98011609(strain Imp.1011-48121) deposited Jan. 16, 1998, accession No. 00012710(strain 1103) and No. 00012709 (strain 1121) deposited Feb. 2, 2000, oran immunogenic or vaccine composition comprised of porcine circovirusproduced on, and isolated from cell culture in vitro, these cells havingbeen infected with a porcine circovirus capable of being isolated from aphysiological sample or from a tissue sample, especially lesions, from apig having the PMWS syndrome, e.g., such a composition wherein theporcine circovirus is produced on, and isolated from a pig kidney cellline, for instance, produced on, and isolated from PK/15 cells free fromcontamination with PCV-1; or such a composition comprising or preparedfrom a culture extract or supernatant, collected from a cell culture invitro which have been infected with a such a circovirus. Thus, porcinecircovirus can be an immunogen. For instance, the vaccine or immunogeniccomposition can comprise the attenuated live whole immunogen (e.g.,virus), advantageously, in a veterinarily or pharmaceutically acceptablevehicle or diluent and optionally an a veterinarily or pharmaceuticallyacceptable adjuvant, as well as, optionally, a freeze-drying stabilizer.The immunogen (e.g., virus) can be inactivated and the vaccine orimmunogenic composition can additional and/or optionally comprise, aveterinarily or pharmaceutically acceptable vehicle or diluent andoptionally a veterinarily or pharmaceutically acceptable adjuvant. Thevaccine or immunogenic composition can comprise PCV-2 immunogens and/orimmunogens of several porcine circoviruses (including PCV-2 or severalstrains of PCV-2, and including PCV-1), as well as optionallyadditionally immunogens from another pig pathogen; e.g, PRRS, Mycoplasmahyopneumoniae, Actinobacillus pleuropneumoniae, E. coli, Pseudorabies,Hog cholera, Bordetella bronchiseptica, Pasteurella multocida,Erysipelothrix rhusiopathiae, Swine Influenza, PPV (see also U.S.application Ser. No. 09/347,594, filed Jul. 1, 1999 and Frenchapplication No. 98 08777, filed Jul. 6, 1998).

For the production of circovirus antigenic preparations, thecircoviruses may be obtained after passage on cells, in particular celllines, e.g. PK/15 cells. The culture supernatants or extracts,optionally purified by standard techniques, may be used.

In the context of attenuated PCV, the attenuation may be carried outaccording to the customary methods, e.g. by passage on cells, preferablyby passage on pig cells, especially cell lines, such as PK/15 cells (forexample from 20 to 150, especially of the order of 40 to 100, passages).

In the context of inactivated vaccine, the PCV, with the fractions whichmay be present, is inactivated according to techniques known to personsskilled in the art. The inactivation will be preferably carried out bythe chemical route, e.g. by exposing the antigen to a chemical agentsuch as formaldehyde (formalin), paraformaldehyde, β-propiolactone orethyleneimine or its derivatives, and/or by physical treatment. Thepreferred method of inactivation will be herein the exposure to achemical agent and in particular to ethyleneimine or to β-propiolactone.

The immunogen in the vaccine or immunogenic composition can be expressedfrom a DNA fragment containing a sequence or fragment thereof(advantageously encoding at least one epitope) selected from the groupconsisting of the sequences designated by the references SEQ ID No: 1,SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 6 (in U.S.application Ser. No. 09/161,092, filed Sep. 25, 1998, U.S. applicationSer. No. 09/082,558, filed May 21, 1998, French applications Nos. 9712382, 98 00873 and 98 03707, filed Oct. 3, 1997, Jan. 22, 1998 and Mar.20, 1998, respectively, and, WO-A-99 18214), as well as SEQ ID No: 7 andSEQ ID No: 8, (FIGS. 1-8). The immunogen in the vaccine or immunogeniccomposition can be expressed from a DNA fragment containing an ORFselected from the group consisting of ORFs 1 to 13, such as ORFs 4, 7,10 and 13; preferably ORFs 4 and/or 13, of a PCV-2 strain, in particularof any one of the above identified strains (as designated in U.S.application Ser. No. 09/161,092, filed Sep. 25, 1998, U.S. applicationSer. No. 09/082,558, filed May 21, 1998, French applications Nos. 9712382, 98 00873 and 98 03707, filed Oct. 3, 1997, Jan. 22, 1998 and Mar.20, 1998, respectively, and, WO-A-99 18214). Thus, the immunogen or aportion thereof, such as an epitope of interest can be obtained by invitro expression thereof from a recombinant or a vector. The immunogenmay be further purified and/or concentrated by the conventional methods.

The immunogen in the vaccine or immunogenic composition can be expressedin vivo by an expression vector comprising a DNA fragment containing asequence or fragment thereof (advantageously encoding at least oneepitope) selected from the group consisting of the sequences designatedby the references SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No:4, SEQ ID No: 6 (in U.S. application Ser. No. 09/161,092, filed Sep. 25,1998, U.S. application Ser. No.09/082,558, filed May 21, 1998, Frenchapplications Nos. 97 12382, 98 00873 and 98 03707, filed Oct. 3, 1997,Jan. 22, 1998 and Mar. 20, 1998, respectively, and, WO-A-99 18214), aswell as SEQ ID No: 7 and SEQ ID No: 8, (FIGS. 1-8). Similarly, theimmunogen in the vaccine, immunogenic or immunological composition canbe expressed in vivo by an expression vector comprising a DNA fragmentcontaining an ORF selected from the group consisting of ORFs 1 to 13,such as ORFs 4, 7, 10 and 13; preferably ORFs 4 and/or 13, of a PCV-2strain, in particular of any one of the above identified strains (asdesignated in U.S. application Ser. No. 09/161,092, filed Sep. 25, 1998,U.S. application Ser. No. 09/082,558, filed May 21, 1998, Frenchapplications Nos. 97 12382, 98 00873 and 98 03707, filed Oct. 3, 1997,Jan. 22, 1998 and Mar. 20, 1998, respectively, and, WO-A-99 18214). Thatis, the vaccine or immunogenic composition can comprise and expressionvector that expresses the immunogen or a portion thereof, e.g., anepitope of interest, in vivo.

The expression vector can be any suitable vector such as a vectorselected from DNA plasmids, bacteria such as E. coli, viruses such asbaculovirus, herpesvirus such as Aujeszky's disease virus, adenovirusincluding porcine adenovirus, poxviruses, especially vaccinia virus,avipox virus, canarypox virus, and swinepox virus, inter a/ia (See alsothe U.S. applications of Audonnet et al. and Bublot et al., Ser. Nos.60/138,352 and 60/138,478, respectively, both filed Jun. 10, 1999 (“DNAVACCINE-PCV”, and “PORCINE CIRCOVIRUS RECOMBINANT POXVIRUS VACCINE”,respectively).

Accordingly, the invention also comprehends nucleic acid molecules andvectors containing them, as well as expression products therefrom,compositions comprising such nucleic acid molecules and/or vectorsand/or expression products, as well as methods for making and using anyor all of these embodiments. The invention especially encompasses hereindisclosed nucleic acid molecules, nucleic acid molecules of documentscited or referenced herein, including PCT WO 99/29717, fragmentsthereof, e.g., ORFs and/or fragments encoding an immunogen or epitope,as well as nucleic acid molecules of strains 1103 and/or 1121, andfragments thereof, as well as vectors comprising these nucleic acidmolecules, compositions comprising these nucleic molecules, vectors, orexpression products therefrom, compositions comprising such expressionproducts, primers or probes for such nucleic acid molecules, and uses ormethods involving these embodiments, e.g., for detecting, diagnosing,assaying for PCV-2, for inducing an immunologenic or protectiveresponse, and the like. Indeed, this invention encompasses anyinventions disclosed and/or claimed in PCT WO 99/29717 or any Nationalapplication claiming priority therefrom or from the U.S. Provisionalsfrom which that PCT claims priority.

As earlier mentioned, embodiments of the invention can includeantibodies. Such antibodies can be polyclonal or monoclonal antibodies;for instance, prepared from the aforementioned circovirus, or from apolypeptide encoded by a DNA fragment having a sequence selected fromthe group consisting of SEQ ID NOS. 1, 2, 3, 4, 6, 7 and 8, (FIGS. 1-8)or from a polypeptide from expression by a vector comprising a sequenceselected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 6, 7 and8; or from a polypeptide from expression by a vector comprising DNAincluding an ORF selected from the group consisting of ORFs 1 to 13 (asdesignated in U.S. application Ser. No. 09/161,092, filed Sep. 25, 1998,U.S. application Ser. No. 09/082,558, filed May 21, 1998, Frenchapplications Nos. 97 12382, 98 00873 and 98 03707, filed Oct. 3, 1997,Jan. 22, 1998 and Mar. 20, 1998, respectively, and, WO-A-99 18214). Theskilled artisan may use techniques known in the art to elicit antibodiesand to generate monoclonal or polyclonal antibodies. Antibodies andantigens can be used in diagnostics.

U.S. application Ser. No. 09/161,092, filed Sep. 25, 1998, U.S.application Ser. No. 09/082,558, filed May 21, 1998, French applicationsNos. 97 12382, 98 00873 and 98 03707, filed Oct. 3, 1997, Jan. 22, 1998and Mar. 20, 1998, respectively, and, WO-A-99 18214 also provide forprobes or primers which can be useful, for instance, in detecting PCV-2DNA, as well as for amplifying PCV-2 DNA, e.g., for preparing anexpression vector. A probe or primer can be any stretch of at least 8,preferably at least 10, more preferably at least 12, 13, 14, or 15, suchas at least 20, e.g., at least 23 or 25, for instance at least 27 or 30nucleotides in PCV-2 genome or a PCV-2 gene which are unique to PCV-2 orwhich are in PCV-2 and are least conserved among the PCV or circovirusfamily. As to PCR or hybridization primers or probes and optimal lengthstherefor, reference is also made to Kajimura et al., GATA 7(4):71-79(1990). Hybridization is advantageously under conditions of highstringency, as the term “high stringency” would be understood by thosewith skill in the art (see, for example, Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. and Hames and Higgins, eds., 1985,Nucleic Acid Hybridization, IRL Press, Oxford, U.K.). Hybridization willbe understood to be accomplished using well-established techniques,including but not limited to Southern blot hybridization, Northern blothybridization, in situ hybridization and, advantageously, Southernhybridization to PCR-amplified DNA fragments.

Like probes or primers, peptides which are not full-length PCV-2proteins are part of invention and can be any stretch of at least 8,preferably at least 10, more preferably at least 12, 13, 14, or 15, suchas at least 20, e.g., at least 23 or 25, for instance at least 27 or 30amino acids in PCV-2 which are unique to PCV-2 or which are in PCV-2 andare least conserved among the PCV and/or circovirus family.Alternatively or additionally, the amino acids of the invention whichare not full length PCV-2 proteins can be an epitopic region of a PCV-2protein.

And, as to DNA and protein sequences used in the invention, they canhave homology, identity or similarity and degrees thereof as defined inU.S. application Ser. No. 09/347,594, filed Jul. 1, 1999 with homology,identity or similarity advantageously determined as discussed in U.S.Ser. No. 09/347,594.

The PCV-2 sequences derived from Meehan et al., 1998 (Strain Imp.1010;ORF1 nucleotides 398-1342; ORF2 nucleotides 1381-314; and correspondrespectively to ORF4 and ORF13 in U.S. application Ser. No. 09/161,092of Sep. 25, 1998 and to COL4 and COL13 in WO-A-9918214). Several PCV-2strains and their sequences are disclosed herein and called Imp 1008,Imp 999, Imp 1011-48285, Imp 1011-48121, 1103 and 1121. Other strainsare disclosed in A. L. Hamel et al. J. Virol. June 1998, vol 72, 6:5262-5267 (GenBank AF027217) and in I. Morozov et al. J. ClinicalMicrob. September 1998 vol. 36, 9: 2535-2541, as well as GenBankAF086834, AF086835 and AF086836. These sequences, or ORFs therefrom, orregions thereof encoding an antigen or immunogen or epitope of interestcan also be used in the practice of this invention.

The invention also encompasses the equivalent sequences to those used ormentioned herein and in documents cited herein; for instance, sequencesthat are capable of hybridizing to the nucleotide sequence under highstringency conditions (see, e.g., Sambrook et al. (1989). Among theequivalent sequences, there may also be mentioned the gene fragmentsconserving the immunogenicity of the complete sequence, e.g., an epitopeof interest.

The homology between the whole genome of PCV types 1 and 2 is about 75%.But within type 2, homology is generally above 95%. Thus, in thepractice of the invention, use of any PCV-2 strain is encompassed byequivalence. A criteria can be that the strain is of type 2, e.g. thathomology at the nucleotide level of the whole genome is equal or greaterthan 85%, advantageously 90% or greater, more advantageously 95% orgreater, preferably 97, 98 or 99% or greater, with the strains disclosedherein, e.g. strain Imp 1010.

Limits of the ORFs of strain Imp 1010 are given in the following table:

Protein size Size of the ORF (amino Name Start End Strand (nucleotides(nt)) acids (aa)) ORF1 103  210 Sense 108 nt 35 aa ORF2 1180  1317 Sense138 nt 45 aa ORF3 1363  1524 Sense 162 nt 53 aa ORF4 398 1342 Sense 945nt 314 aa  ORF5 900 1079 Sense 180 nt 59 aa ORF6 1254  1334 Sense  81 nt26 aa ORF7 1018   704 Antisense 315 nt 104 aa  ORF8 439  311 Antisense129 nt 42 aa ORF9 190  101 Antisense  90 nt 29 aa ORF10 912  733Antisense 180 nt 59 aa ORF11 645  565 Antisense  81 nt 26 aa ORF12 1100 1035 Antisense  66 nt 21 aa ORF13 314 1381 Antisense 702 nt 213 aa 

The ORFs are defined with respect to strain Imp 1010. The invention alsoencompasses the use of the corresponding ORFs in any other PCV-2 strain,and any of the PCV-2 strains as defined herein or in documents citedherein. Thus, from the genomic nucleotide sequence, it is routine art todetermine the ORFs using a standard software, such as MacVector®. Also,alignment of genomes with that of strain 1010 and comparison with strain1010 ORFs allows the one skilled in the art to readily determine theORFs on the genome for another strain (e.g. those disclosed in WO-A-9918214). Using software or making alignment is not undue experimentationand directly provides access to equivalent ORFs.

Also equivalent and useful in the practice of the invention are thenucleotide sequences which change neither the functionality nor thestrain specificity (say of strain type 2) of the gene considered orthose of the polypeptides encoded by this gene. The sequcnces differingthrough the degeneracy of the code are, of course, be included in thepractice of the invention.

For ORF4, homology between PCV-1 and PCV-2 is about 86%, and for ORF13,the homology between PCV-1 and PCV-2 is about 66%. Thus, also equivalentsequences useful in the practice of the present invention, for ORF4, arethose sequences having an homology equal or greater than 88%,advantageously 90% or greater, preferably 92% or 95% or greater homologywith OPF4 of strain Imp 1010, and for ORF13, those sequences having anhomology equal or greater than 80%, advantageously 85% or greater,preferably 90% or 95% or greater than ORF13 of strain Imp 1010. (Usingthe terminology of U.S. application Ser. No. 09/161,092 of Sep. 25,1998.)

For homology regarding the other ORFs, one can determine those sequenceswhich come from a PCV strain having an ORF4 and/or an ORF13 which havean homology as defined above with the corresponding ORF of strain 1010.For ORF7, sequences useful in the practice of the invention includethose sequences having an homology that is advantageously equal to orgreater than 80%, more advantageously 85% or greater, preferably 90% or95% or greater with ORF7 of strain Imp 1010. For ORF10, sequences usefulin the practice of the invention include those sequences having anhomology that is advantageously equal to or greater than 86%, moreadvantageously 90% or greater, preferably 95% or greater with ORF10 ofstrain Imp 1010. (Using the terminology of U.S. application Ser. No.09/161,092 of Sep. 25, 1998.)

Also, equivalent sequences useful in the practice of this presentinvention, for ORF1 of Meehan et al., 1998, are those sequences havingan homology equal or greater than 88%, advantageously 90% or greater,preferably 92% or 95% or greater with ORF1 of strain Imp 1010, and forORF2 of Meehan et al., 1998, are those sequences having an homologyequal or greater than 80%, advantageously 85% or greater, preferably 90%or 95% or greater with ORF2 of strain Imp 1010.

ORF1 and ORF2 according to Meehan et al., 1998 has the potential toencode proteins with predicted molecular weights of 37.7 kD and 27.8 kDrespectively. ORF3 and ORF4 (according to Meehan et al. 1998, correspondto ORF7 and ORF10 respectively in WO-A-9918214 and/or U.S. applicationSer. No. 09/161,092 of Sep. 25, 1998) has the potential to encodeproteins with predicted molecular weights of 11.9 and 6.5 kDrespectively. The sequence of these ORFs is also available in Genbank AF055392. They can also be incorporated in plasmids and be used inaccordance with the invention alone or in combination, e.g. with ORF1and/or ORF2 of Meehan et al., 1998.

The other ORFs 1-3 and 5, 6, 8-9, 11-12 disclosed in U.S. applicationSer. No. 09/161,092 of Sep. 25, 1998 (COLs 1-3 and 5, 6, 8-9, 11-12 inWO-A-9918214), or region(s) thereof encoding an antigen or epitope ofinterest, may be used in the practice of this invention, e.g., alone orin combination or otherwise with each other or with the ORFs 1 and/or 2of Meehan et al., 1998 or region(s) thereof encoding antigen(s) orepitope(s). Similarly, for homology, one can determine that there areequivalent sequences which come from a PCV strain having an ORF2 and/oran ORF1 which have an homology as defined above with the correspondingORF of strain 1010 as defined in Meehan et al., 1998. For ORF3 accordingto Meehan et al., 1998, an equivalent sequence has homology thereto thatis advantageously, for instance, equal or greater than 80%, for example85% or greater, preferably 90% or 95% or greater with ORF3 of strain Imp1010. And, for ORF4 according to Meehan et al., 1998, advantageously anequivalent sequence has homology that is equal or greater than 86%,advantageously 90% or greater, preferably than 95% or greater with ORF4of strain Imp 1010.

Nucleotide sequence homology can be determined using the “Align” programof Myers and Miller, (“Optimal Alignments in Linear Space”, CABIOS 4,11-17, 1988, incorporated herein by reference) and available at NCBI.Alternatively or additionally, the term “homology” or “identity”, forinstance, with respect to a nucleotide or amino acid sequence, canindicate a quantitative measure of homology between two sequences. Thepercent sequence homology can be calculated as(N_(ref)−N_(dif))*100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence similarity of 75% with thesequence AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” or “identity” with respect tosequences can refer to the number of positions with identicalnucleotides or amino acids divided by the number of nucleotides or aminoacids in the shorter of the two sequences wherein alignment of the twosequences can be determined in accordance with the Wilbur and Lipmanalgorithm (Wilbur and Lipman. 1983 PNAS USA 80:726, incorporated hereinby reference), for instance, using a window size of 20 nucleotides, aword length of 4 nucleotides, and a gap penalty of 4, andcomputer-assisted analysis and interpretation of the sequence dataincluding alignment can be conveniently performed using commerciallyavailable programs (e.g., Intelligenetics ™ Suite, Intelligenetics Inc.Calif.). When RNA sequences are said to be similar, or have a degree ofsequence identity or homology with DNA sequences, thymidine (T) in theDNA sequence is considered equal to uracil (U) in the RNA sequence.

RNA sequences within the scope of the invention can be derived from DNAsequences, by thymidine (T) in the DNA sequence being considered equalto uracil (U) in RNA sequences.

Additionally or alternatively, amino acid sequence similarity oridentity or homology can be determined using the BlastP program(Altschul et al., Nucl. Acids Res. 25, 3389-3402, incorporated herein byreference) and available at NCBI. The following references (eachincorporated herein by reference) provide algorithms for comparing therelative identity or homology of amino acid residues of two proteins,and additionally or alternatively with respect to the foregoing, theteachings in these references can be used for determining percenthomology or identity: Needleman S B and Wunsch C D, “A general methodapplicable to the search for similarities in the amino acid sequences oftwo proteins,” J. Mol. Biol. 48:444-453 (1970); Smith T F and WatermnanM S, “Comparison of Bio-sequences,” Advances in Applied Mathematics2:482-489 (1981); Smith T F, Waterman M S and Sadler J R, “Statisticalcharacterization of nucleic acid sequence functional domains,” NucleicAcids Res., 11:2205-2220 (1983); Feng D F and Dolittle R F, “Progressivesequence alignment as a prerequisite to correct phylogenetic trees,” J.of Molec. Evol., 25:351-360 (1987); Higgins D G and Sharp P M, “Fast andsensitive multiple sequence alignment on a microcomputer,” CABIOS, 5:151-153 (1989); Thompson J D, Higgins D G and Gibson T J, “ClusterW:improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighing, positions-specific gap penalties and weightmatrix choice, Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J,Haeberlie P and Smithies O. “A comprehensive set of sequence analysisprogram for the VAX,” Nucl. Acids Res., 12: 387-395 (1984).

The invention further comprehends uses of a PCV-2 immunogen, eitheralone or in further combination with an immunogen of another porcinepathogen to generate compositions according to the invention, e.g.,admixing the ingredients; and, the invention also therefore comprehendskits wherein components are individually contained and optionally thecontainers are packaged together for admixture and/or administration,wherein the kit can also optionally include instructions for admixtureand/or administration.

While the invention has been discussed in terms of administering tofemale pigs immunogenic or vaccine compositions comprising a PCV-2immunogen, the invention can also comprehend administering suchcompositions to sow or gilt and/or to boar as described herein; Thus,both mother and offspring (e.g., sow, gilt) and boar can be administeredcompositions of the invention and/or can be the subject of performanceof methods of the invention. Accordingly, populations of pigs can beadministered compositions of the inventions and/or can be the subject ofperformance of methods of the invention.

According to the present invention, immunogenic and vaccine compositionsmay comprise immunogens from more than one PCV-2 strain. For example, itis possible to combine immunogens from strains 1121 and 1103, from oneor both of these strains with at least one other strain disclosedherein, or any other combination.

The present invention provides for methods allowing the one skilled inthe art to evaluate the efficacy of vaccines against PCV-2. A firstmethod is an ELISA method or with seroneutralization. A second method isa vaccination followed by challenge with a virulent PCV-2 strain, e.g.one of the strains disclosed herein. In other words, the inventionallows one to check for PCV immunogens, including PCV-1 immunogens ableto elicit an immunologenic or protective response against PCV-2.

Thus one aspect of the invention is to provide immunogenic or vaccinalcompositions comprising a PCV immunogen and able to elicit animmunogenic or protective response against PCV-2. The invention relatesalso to methods of immunization or vaccination using such an immunogen,as well as to the use of such an immunogen to produce such animmunogenic or vaccinal composition.

The invention shall be further described by way of the following ExampleAnd Results, provided for illustration and not to be considered alimitation of the invention.

EXAMPLE AND RESULTS Example/Result 1 Myocarditis, Abortion andIntrauterine Infection Associated with PCV-2

Late term abortions and farrowings with both stillborn and mummifiedpiglets occurred in a new 450-female pig swine facility as it wasbrought into production. Pseudopregnancy was also observed in severalgilts. Gilts received two doses of an inactivated vaccine containingparvovirus and leptospiral immunogens prior to breeding.

A litter received for postmortem examination consisted of nine fetusesthat appeared to have died at various stages of gestation. There were 2mummified, 2 macerated, 3 autolysed and 2 fresh, stillborn piglets.Lesions were observed on gross pathological examination in one partiallyautolysed fetus only. In this fetus both ventricles of the heart weredilated, the liver was enlarged and firm and there was both hydrothoraxand ascites. Histopathologically, there were extensive areas ofmyocardial degeneration or necrosis with edema and mild fibrosis, and adiffuse moderate -infiltration of lymphocytes and macrophages. There wasmarked generalized hepatic congestion and hepatocellular loss. Thespleen and kidneys were also congested. Significant histological lesionswere not detected in the other fetuses.

Immunohistochemical staining for PCV-2 was performed as previouslydescribed using a rabbit polyclonal antiserum and a monoclonal antibodythat were raised against PCV-2. on sections of formalin-fixed, routinelyprocessed and embedded tissue (Ellis et al., 1998; Ellis et al., 1999).In the fetus with dilated cardiomyopathy there was extensive stainingfor PCV-2 antigen throughout the affected myocardium. Staining was mostextensive in areas of necrosis and appeared to involve primarilymyocytes. Both cytoplasmic and nuclear staining was present. In multiplefetuses there was extensive staining in the liver. In some sections itappeared to involve primarily sinusoidal endothelium and Kupfer cells,while in other fetuses, including the one with myocarditis, there wasalso nuclear and cytoplasmic staining of hepatocytes. Positively stainedcells were scattered throughout the lung, and multifocally in thekidney. Polymerase chain reaction for PCV-2 was performed as previouslydescribed using frozen tissue (Ellis et al., 1999). PCR product of theexpected size for PCV-2 was amplified from fetal tissue. PCV-2 wasisolated from the fetus with myocarditis and a pool of tissues fromother fetuses in the litter by inoculating tissue homogenates ontoPCV-free PK-15 cells.

Fetal tissues were also examined for other viral pathogens that havebeen associated with fetal injury and abortions in swine, including,porcine parvovirus (PPV), porcine reproductive and respiratory syndromevirus (PRRSV), encephalomyocarditis (EMCV), and enteroviruses. PPVantigen was not detected by fluorescent antibody testing (FAT) on frozensections of lung, liver, and spleen from the mummified or stillbornfetuses. Homogenates of liver, lung, and spleen from the aborted fetuseswere also inoculated into cultures of PCV-free PK-15 cells, primaryporcine fallopian tube cells and Vero cells. Cytopathic viruses were notdetected after three passages. Tissues were negative for PPV using PCR.PRRSV antigen was not detected by immunohistochemical staining.

Thus, there were fetal lesions and abortion directly associated withPCV-2. These results also show vertical transmission of the virus.

In a previous study, PCV-1 was isolated from 2 of 160 pig fetusesexamined, implying that this group of viruses can be verticallytransmitted; however, PCV-1 antigen could not be associated with anylesions in the tissue (Allan et al., 1995). The exclusion of otheragents that have been associated with fetal lesions and abortion inswine, including, PPV (Bolt et al., 1997; Molitor et al., 1991), PRRSV(Lager et al., 1996), EMCV (Kim et al., 1989), and enterovirus (Molitoret al., 1991) indicate that PCV-2 can cause significant fetal pathologyand subsequent abortion.

However, PCV-1 immunogens (still according to the general definitiongiven at the beginning) may elicit an immunogenic or protective responseagainst myocarditis and/or abortion and/or intrauterine infection aswell as post-weaning multisystemic wasting syndrome and ergo PCV-1immunogens can also be used in the practice of this invention (e.g., inthe methods, compositions, uses, etc.)—either alone or in conjunctionwith PCV-2 immunogens (the vector can contain and express DNA encodingfor both a PCV-1 immunogen and/or epitope and a PCV-2 immunogen and/orepitope) and/or alone or in conjunction an immunogen and/or epitope ofother porcine pathogen (if a vector is used, the vector can contain andexpress DNA encoding for both a PCV-1 immunogen and/or epitope and animmunogen and/or epitope of another porcine pathogen, or for a PCV-1immunogen and/or epitope and a PCV-2 immunogen and/or epitope and animmunogen and/or epitope of another porcine pathogen). Thus, one skilledin the art may alternatively or additionally use a PCV-1 immunogen,and/or epitope and/or vector encoding such an immunogen and/or epitopein the practice of this invention without any undue experimentation; forinstance, to so do, one need only read the text herein prior to thisExample and at the conclusion of (after) this Example, andsubstitute—PCV-1—for “PCV-2” with any modification minor based onteachings herein.

The wasting syndrome associated with PCV-2 infection most often occursin 5-12 week old pigs (Allan et al., 1998; Ellis et al., 1998).Experimental infection of neonatal swine indicates a relatively longprodromal period between infection and the development of clinical signsassociated with PCV-2 (Allan et al. 1999; Ellis et al. 1999). Thefindings herein show that the virus is transmitted vertically or in theperinatal period. Not only may interuterine vertical transmission ofPCV-2 result in abortion, but it is possible that sublethally inutero-infected piglets may be the animals that subsequently developPMWS.

Furthermore, these results show that inoculation of female pigs with acomposition comprising an PCV-2 immunogen (which composition can alsoinclude an immunogen from another porcine pathogen, e.g., porcineparvovirus), prior to breeding or serving, or prior to the perinatalperiod and/or during gestation can prevent myocarditis and/or abortionand/or intrauterine infection associated with porcine circovirus-2, aswell as post-weaning multisystemic wasting syndrome and other pathologicsequelae associated with PCV-2, by eliciting an immunological responseor antibodies against PCV-2.

Of course, compositions, methods, and other aspects of the invention canbe used or practiced in animals other than pigs, e.g., sheep, bison,cattle, wild boar; for instance, if PCV-2 infects such other animals.

Example/Result 2 Myocarditis, Abortion and Intrauterine InfectionAssociated with PCV-2

The presence of PCV-2 in neonatal piglets suggests that verticaltransmission may be an important means of viral transmission. This modeof transmission may be related not only to reproductive failure, butalso to the development of multisystemic disease later in life. It is ofinterest to determine whether previously undetected PCV-2 (and PCV-1)has been vertically transmitted in pork producing areas where PMWS, andby extension PCV-2 infection, has been endemic for at least severalyears.

Thirty eight submissions involving reproductive failure received in thediagnostic laboratory at the Western College of Veterinary Medicine(WCVM), University of Saskatchewan, Saskatoon, Canada, over a four-yearperiod from a total of 30 high health herds in Canada were evaluated.Five of the farms from which the samples were obtained had diagnosedcases of PMWS. Twenty-seven of the thirty-eight submissions (71%) wereclassified as abortions; five of these (13%) also involved at least onemummified fetus. Of the remaining 10 cases: 5 involved stillborn pigletsalong, with nonviable piglets (13%); 2 with stillborn and one or moremummified feti (5%); 2 with only stillborn piglets (5%); and one withonly mummified feti (2.5%). Routine diagnostics for pathogens other thancircovirus revealed 4 cases (11%) in which the etiology was determinedto be porcine parvovirus and 2 cases (5%) in which the etiology wasdetermined to be of bacterial origin. Gross necropsies were performedand tissues were collected and fixed in buffered formalin (fixation time24-72 hrs) and, in most cases, fresh tissues were also submitted forroutine microbiological evaluation. None of these cases had beenpreviously tested for PCV-2.

The PCR technique used for the detection of PCV-1 and PCV-2 wasperformed as previously described (Tischer et al. 1974). PCV-1 was notdetected by PCR in any submissions comprising reproductive failure fromthe four-year period. PCV-2 was detected by PCR in two differentsubmissions that originated from the same multi-site pork productionunit on two separate occasions in the spring of the last year in thefour-year period. The first of these submissions comprised a litter ofpiglets with gross evidence of myocarditis, cardiac hypertrophy, andchronic passive congestion.

Immunohistochemical identification of PCV-2 in tissues was performed aspreviously described (Tischer et al. 1974). Immunohistochemical staining(IHC) for PCV-2 was positive in hearts from all six of the piglets thatwere submitted, while 4 of 6 were positive by PCV-2 PCR (see followingTable).

Table: Detection of PCV-2 in the formalin fixed hearts of porcine withmyocarditis by PCR, IHC and viral isolation in cell culture.

PCV-2 postive tissues PCR IHC Virus Isolation Fixed 5/6 6/6 N/A Frozen4/4 N/A 2/4

The second submission from the same farm consisted of a litter of fourpiglets in which 2 were stillborn and 2 others died shortly after birth.All four piglets also had gross evidence of a severe, difusemyocarditis, cardiac hypertrophy, and chronic passive congestion. Onlyfresh frozen heart, and pooled lung/spleen tissues were submitted foranalysis. PCV-2 PCR was positive in the hearts of 2 of 4 piglets and inthe pooled lung and splenic tissues of 4 of 4 piglets. Isolation ofPCV-2 from affected hearts and/or pooled lung and splenic tissue waspositive in 2 of the 4 cases that were PCV-2 positive by PCR. Based onserology and/or PCR, other agents associated with reproductive failurein swine, including porcine reproductive and respiratory syndrome virusand porcine parvovirus were apparently circulating in the breeding herd.However, these agents could not be shown to be associated with thesevere cardiac (or other) lesions in the affected piglets; but, they maycontribute to PMWS.

PCV-2 was not detected by PCR or IHC in any representative cases ofreproductive failure submitted during the first three years of thefour-year period (it was detected in cases of reproductive failuresubmitted during the last year of the four-year period). In order torule out damage to DNA due to formalin fixation as a possible adversefactor limiting the ability to detect PCV-2 by PCR, PCR was performed ontissues collected from four weanling piglets with PMWS, PCV-2 DNA wasamplified in all fixed tissues tested, including; lung, liver, kidneyand bronchial lymph node, from all four individuals. Moreover, thesensitivity of the PCR PCV-2 was independent of the length of time thateach tissue was fixed in formalin.

These results confirm and extend the previous observation (West et al.1999) that PCV-2 can be vertically transmitted and can be present inlarge amounts within lesions from piglets infected in utero. Verticaltransmission of PCV-2 virus and resultant fetal damage, such asmyocarditis, is an additional disease manifestation of PCV-2.Furthermore, the failure to detect PCV-2 in cases of reproductivefailure prior to the last year of the four-year period from an endemicarea of PCV-2 infection may indicate that vertical transmission was notthe primary mechanism responsible for the initial dissemination of viralinfection. Sexual, as well as vertical, modes of transmission can beattributed to the spread of PCV-2 infection in pigs.

Example/Result 3 Culture and Isolation of the Porcine Circovirus Strains

Viruses 1103 and 1021 were isolated respectively in Alberta,respectively Saskatoon, Canada, from abortive cases according to themethod described in J. Ellis et al. Can. J. Vet. 1998, vol 39,44-51.

Viral culture is carried out on PK/15 cell cultures, known to beuncontaminated with the porcine circovirus (PCV), pestiviruses, porcineadenoviruses and porcine parvoviruses (Allan G. et al Pathogenesis ofporcine circovirus experimental infections of colostrum-deprived pigletsand examination of pig foetal material. Vet. Microbiol. 1995, 44,49-64).

Monolayers of PK/15 cells are dissociated by trypsinization (with atrypsin-versene mixture) from confluent cultures, and taken up in MEM-SAmedium containing 15% foetal calf serum not contaminated by pestivirus(=MEM-G medium) in a final concentration of about 400,000 cells per ml.10 ml aliquot fractions of this cell suspension are then mixed with 2 mlaliquot fractions of the inocula described above, and the final mixturesis aliquoted in 6 ml volumes in two Falcon flasks of 25 cm². Thesecultures are then incubated at +37° C. for 18 hours under an atmospherecontaining 10% CO₂.

After incubation, the culture medium of the semi-confluent monolayerswere treated with 300 mM D-glucosamine (Cat #G48175, Sigma-AldrichCompany Limited, Poole, UK) (Tischr I. et al., Arch. Virol., 1987 9639-57), then incubation was continued for an additional period of 48-72hours at +37° C. Following this last incubation, one of the two Falconsof each inoculum was subjected to 3 successive freeze/thaw cycles. ThePK/15 cells of the remaining Falcon were treated with a trypsin-versenesolution, resuspended in 20 ml of MEM-G medium, and then inoculated into75 cm² Falcons at a concentration of 400,000 cells/ml. The freshlyinoculated flasks were then “superinfected” by addition of 5 ml of thecorresponding lysate obtained after the freeze/thaw cycles.

Example/Result 4 Technique for the Detection of PCV byImmunofluorescence

The initial screening of all the cell culture preparations fixed withacetone was carried out by an indirect immunofluorescence technique(IIF) using a {fraction (1/100)} dilution of a pool of adult pig sera.This pool of sera comprises sera from 25 adult sows from NorthernIreland and is known to contain antibodies against a wide variety ofporcine viruses, including PCV: porcine parvovirus, porcine adenovirus,and PRRS virus. The IIF technique was carried out by bringing the serum(diluted in PBS) into contact with the cell cultures for one hour at+37° C., followed by two washes in PBS. The cell cultures were thenstained with a {fraction (1/80)} dilution in PBS of a rabbit anti-pigimmunoglobulin antibody conjugated with fluorescein isothiocyanate forone hour, and then washed with PBS and mounted in glycerol buffer priorto the microscopic observation under ultraviolet light.

Example/Result 5 Production of PCV Antigens by in Vitro Culture

The culture of the noncontaminated PK/15 cells and the viralmultiplication were carried out according to the same methods as inExample 1. The infected cells are harvested after trypsinization after 4days of incubation at 37° C. and enumerated. The next passage isinoculated with 400,000 infected cells per ml.

The various PCV-2 strains disclosed herein, e.g. strains 1103 and 1121are so cultivated.

Example/Result 6 Titration of PCV-2

Titration is carried out in 96-well microplates. A suspension of PK/15cells (150 000 cells per ml) is first introduced (100 μl per well). Thendilutions of the viral culture are done and 100 μl thereof areintroduced in the wells. Incubation is done at 37° C. with CO₂. After 24h, there is carried out a treatment with glucosamine half an hour at 37°C. (for the conditions see example 3). The culture medium is thenremoved and fresh medium is introduced. Incubation is conducted 72 h at37° C. Revelation of the foci is done using an anti-PCV-2 monoclonalantibody and a FITC labelled mouse conjugate.

This method can be used to titration for preparing inactivated as wellas live attenuated PCV-2.

Example/Result 7 Inactivation of the Viral Antigens

At the end of the viral culture, the infected cells are harvested andlysed using ultrasound (Branson Sonifier) or with the aid of arotor-stator type colloid mill (UltraTurrax, IKA). The suspension isthen centrifuged at 3700 g for 30 minutes. The viral suspension isinactivated with 0.1% ethyleneimine for 18 hours at +37° C. or with 0.5%beta-propiolactone for 24 hours at +28° C. If the virus titre beforeinactivation is inadequate, the viral suspension is concentrated byultrafiltration using a membrane with a 300 kDa cut-off (MilliporePTMK300). The inactivated viral suspension is stored at +5° C.

Example/Result 8 Preparation of the Vaccine in the Form of an EmulsionBased on Mineral Oil

The vaccine is prepared according to the following formula:

suspension of inactivated porcine circovirus: 250 ml

Montanide® ISA 70 (SEPPIC): 750 ml

The aqueous phase and the oily phase arc sterilized separately byfiltration. The emulsion is prepared by mixing and homogenizing theingredients with the aid of a Silverson turbine emulsifier.

One vaccine dose contains about 10^(7.5) TCID₅₀. The volume of onevaccine dose is 0.5 ml for administration by the intradermal route, and2 ml for administration by the intramuscular route.

This vaccine is used in a vaccination programme against themultisystemic wasting syndrome in combination with the Parvovax®vaccine.

Example/Result 9 Preparation of the Vaccine in the Form of aMetabolizable Oil-based Emulsion

The vaccine is prepared according to the following formula:

suspension of inactivated porcine circovirus 200 ml

Dehymuls HRE 7 (Henkel): 60 ml

Radia 7204 (Oleofina): 740 ml

The aqueous phase and the oily phase are sterilized separately byfiltration. The emulsion is prepared by mixing and homogenizing theingredients with the aid of a Silverson turbine emulsifier.

One vaccine dose contains about 10^(7.5) TCID₅₀. The volume of onevaccine dose is 2 ml for administration by the intramuscular route.

This vaccine is used in a vaccination programme against themultisystemic wasting syndrome in combination with the Parvovax®vaccine.

Example/Result 10 Vaccination of Piglets with DNA (Plasmid) Vector

Groups of 3 or 4 piglets, caesarian-derived day 0 are placed intoisolators. The piglets are vaccinated day 2 either with (A) a plasmidcomprising ORF 13 or with (B) a mixture of this plasmid and anotherplasmid comprising ORF 4, and with a physiological solution for thecontrol group. Each plasmid is diluted in sterile physiological solution(NaCl 0,9%) at 250 μg/μl final concentration. A 2 ml volume is injectedby intramuscular route in two points of 1 ml (1 point each side of theneck). A second injection of vaccine or placebo is administered day 14.Vaccination with DNA is well tolerated by piglets and no evidence foradverse reaction to vaccination is noted. The piglets are challenged day21 by oronasal administration of PCV-2 viral suspension, 1 ml in eachnostril. After challenge piglets are weighed once a week. Rectaltemperatures are recorded on days 17, 21, 22, 24, 27, 29, 31, 34, 37,41, 44. Day 44 fecal swabs are collected from each piglet for PCV-2shedding. The virus is detected and quantified by quantitative PCR. Day45 necropsies are performed and tissue samples are collected for virusisolation.

Clinical Symptoms:

There is no significant difference for the mean body weight gains or themean body temperatures between groups.

Necropsy Lesions

The only gross finding noted in pigs at termination is bronchiallymphadenopathy. The lesions are scored according the followingcriteria.

0=no visible enlargement of lymph nodes

1=mild lymph nodes enlargement, restricted to bronchial lymph nodes

2=moderate lymph nodes enlargement, restricted to bronchial lymph nodes

3=severe lymph nodes enlargement, extended to bronchial submandibullarprescapsular and inguinal lymph nodes.

std is an abbreviation for standard deviation

Lymphadenopathy scores Groups mean std N (A) 1.2 1.3 4 (B) 2.0 1.7 3controls 3.0 0.0 3 N = number of piglets in each group

A reduction of the lymph node lesions is observed in 3 out 4 pigletsimmunized with (A) and 1 out 3 piglets immunized with (B) mixture. Thisdifference is not significant (p>0.05) due to the high value of thestandard deviations (std).

Virus Load in Lymph Nodes Tissues:

Quantitative virus re-isolation is performed on tissue homogenatesprepared from bronchial and mesenteric lymph nodes.

The data presented correspond to the virus titers in tissue homogenatesafter transformation in log₁₀.

PCV-2 titers Bronchial LN Mesenteric LN Groups mean std mean std N (A)0.9 0.8 0.9 0.8 4 (B) 0.7 0.6 0.2 0.2 3 Controls 2.0 1.1 1.8 1.1 4

Bronchial lymph nodes seem to contain the most infectious virus. Areduction of the viral load is observed in bronchial and mesentericlymph nodes from piglets immunized with either (A) or (B) mixture. Thisreduction is significant (p #0.05 for the plasmids mixture.

Viral Excretion:

Post challenge fecal swabs are assessed for schedding PCV-2 by PCR basedon amplification of PCV-2 ORF 13. Each assay is performed in triplicateon 2 ml of sample. Unvaccinated controls are negative for PCV-2 priorchallenge and positive after challenge confirming the validity of thePCR assay.

Values are expressed as log₁₀ (number of molecules of PCV-2 in 2 μlsample).

Log₁₀ number of PCV-2 DNA molecules Groups Mean std N (A) 3.3 0.3 4 (B)2.9 0.7 3 Controls 3.6 0.6 4

The differences between groups are not significant (p >0.05).

Example/Result 11 Vaccination of Piglets with Canarypox live Vector andResults

Groups of 3 or 4 piglets, caesarian-derived day 0 are placed intoisolators. Day 2 the piglets are vaccinated with 10⁸ pfu of (C) acanarypox comprising ORF 13, or (D) of a canarypox comprising ORF 13 andORF 4, or parental canarypox, in 1 ml of PBS, by intramuscular route onthe side of the neck. A second injection of vaccine or placebo isadministered at day 14. Vaccination with canarypox is well tolerated bypiglets and no evidence for adverse reaction to vaccination is noted Thepiglets are challenged day 21 by oronasal administration of a PCV-2viral suspension, 1 ml in each nostril. Day 45 necropsies are performedand samples of tissues are collected for virus isolation.

Necropsy Results

PMWS is characterized generally by lymphadenopathy and more rarely byhepatitis or nephritis. So the gross findings in lymph nodes are scoredfor each piglet in the following manner: 0=no visible enlargement oflymph nodes; 1=mild lymph nodes enlargement, restricted to bronchiallymph nodes; 2=moderate lymph nodes enlargement, restricted to bronchiallymph nodes; 3=severe lymph nodes enlargement, extended to bronchial,submandibullar prescapular and inguinal lymph nodes.

Groups Scores (C) 0.5 0.0 0.0 mean 0.38 standard deviation 0.48 (D) 0.00.5 0.5 1.0 mean 0.5 standard deviation 0.41 Controls 2.0 2.5 2.5 2.5mean 2.38 standard deviation 0.25

Bronchial lymphadenopathy for PCV-2 is a prominent gross finding. Asignificant reduction of the lymph nodes lesion in relation to controlgroup is observed after immunization with (C) and (D) (p≦0.05).

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

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11. Molitor T W, Orveerakul K, Zhang Z Z, et al.: 1991, Polymerase chainreaction (PRC) amplification for detection of porcine parvovirus. JVirol Meth 32:201-211

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8 1 1767 DNA Porcine circovirus 1 aattcaacct taacctttct tattctgtagtattcaaagg gcacagagcg ggggtttgag 60 ccccctcctg ggggaagaaa gtcattaatattgaatctca tcatgtccac cgcccaggag 120 ggcgttctga ctgtggttcg cttgacagtatatccgaagg tgcgggagag gcgggtgttg 180 aagatgccat ttttccttct ccagcggtaacggtggcggg ggtggacgag ccaggggcgg 240 cggcggagga tctggccaag atggctgcgggggcggtgtc ttcttctccg gtaacgcctc 300 cttggatacg tcatatctga aaacgaaagaagtgcgctgt aagtattacc agcgcacttc 360 ggcagcggca gcacctcggc agcacctcagcagcaacatg ccgagcaaga agaatggaag 420 aagcggaccc caaccccata aaaggtgggtgttcactctg aataatcctt ccgaagacga 480 gcgcaagaaa atacgggatc ttccaatatccctatttgat tattttattg ttggcgagga 540 gggtaatgag gaaggacgaa cacctcacctccaggggttc gctaattttg tgaagaagca 600 gacttttaat aaagtgaagt ggtatttgggtgcccgctgc cacatcgaga aagcgaaagg 660 aacagatcag cagaataaag aatactgcagtaaagaaggc aacttactga tggagtgtgg 720 agctcctaga tctcagggac aacggagtgacctgtctact gctgtgagta ccttgttgga 780 gagcgggagt ctggtgaccg ttgcagagcagcaccctgta acgtttgtca gaaatttccg 840 cgggctggct gaacttttga aagtgagcgggaaaatgcag aagcgtgatt ggaagactaa 900 tgtacacgtc attgtggggc cacctgggtgtggtaaaagc aaatgggctg ctaattttgc 960 agacccggaa accacatact ggaaaccacctagaaacaag tggtgggatg gttaccatgg 1020 tgaagaagtg gttgttattg atgacttttatggctggctg ccctgggatg atctactgag 1080 actgtgtgat cgatatccat tgactgtagagactaaaggt ggaactgtac cttttttggc 1140 ccgcagtatt ctgattacca gcaatcagaccccgttggaa tggtactcct caactgctgt 1200 cccagctgta gaagctcttt atcggaggattacttccttg gtattttgga agaatgctac 1260 agaacaatcc acggaggaag ggggccagttcgtcaccctt tcccccccat gccctgaatt 1320 tccatatgaa ataaattact gagtcttttttatcacttcg taatggtttt tattattcat 1380 taagggttaa gtggggggtc tttaagattaaattctctga attgtacata catggttaca 1440 cggatattgt attcctggtc gtatatactgttttcgaacg cagtgccgag gcctacgtgg 1500 tctacatttc cagcagtttg tagtctcagccacagctggt ttcttttgtt gtttggttgg 1560 aagtaatcaa tagtggaatc taggacaggtttgggggtaa agtagcggga gtggtaggag 1620 aagggctggg ttatggtatg gcgggaggagtagtttacat aggggtcata ggtgagggct 1680 gtggcctttg ttacaaagtt atcatctagaataacagcac tggagcccac tcccctgtca 1740 ccctgggtga tcggggagca gggccag 17672 1767 DNA Porcine circovirus 2 aattcaacct taacctttct tattctgtagtattcaaagg gcacagagcg ggggtttgag 60 ccccctcctg ggggaagaaa gtcattaatattgaatctca tcatgtccac cgcccaggag 120 ggcgttttga ctgtggttcg cttgacagtatatccgaagg tgcgggagag gcgggtgttg 180 aagatgccat ttttccttct ccagcggtaacggtggcggg ggtggacgag ccaggggcgg 240 cggcggagga tctggccaag atggctgcgggggcggtgtc ttcttctccg gtaacgcctc 300 cttggatacg tcatatctga aaacgaaagaagtgcgctgt aagtattacc agcgcacttc 360 ggcagcggca gcacctcggc agcacctcagcagcaacatg cccagcaaga agaatggaag 420 aagcggaccc caaccccata aaaggtgggtgttcactctg aataatcctt ccgaagacga 480 gcgcaagaaa atacgggatc ttccaatatccctatttgat tattttattg ttggcgagga 540 gggtaatgag gaaggacgaa cacctcacctccaggggttc gctaattttg tgaagaagca 600 gacttttaat aaagtgaagt ggtatttgggtgcccgctgc cacatcgaga aagcgaaagg 660 aacagatcag cagaataaag aatactgcagtaaagaaggc aacttactga tggagtgtgg 720 agctcctaga tctcagggac aacggagtgacctgtctact gctgtgagta ccttgttgga 780 gagcgggagt ctggtgaccg ttgcagagcagcaccctgta acgtttgtca gaaatttccg 840 cgggctggct gaacttttga aagtgagcgggaaaatgcag aagcgtgatt ggaagactaa 900 tgtacacgtc attgtggggc cacctgggtgtggtaaaagc aaatgggctg ctaattttgc 960 agacccggaa accacatact ggaaaccacctagaaacaag tggtgggatg gttaccatgg 1020 tgaagaagtg gttgttattg atgacttttatggctggctg ccctgggatg atctactgag 1080 actgtgtgat cgatatccat tgactgtagagactaaaggt ggaactgtac cttttttggc 1140 ccgcagtatt ctgattacca gcaatcagaccccgttggaa tggtactcct caactgctgt 1200 cccagctgta gaagctcttt atcggaggattacttccttg gtattttgga agaatgctac 1260 agaacaatcc acggaggaag ggggccagttcgtcaccctt tcccccccat gccctgaatt 1320 tccatatgaa ataaattact gagtcttttttatcacttcg taatggtttt tattattcat 1380 taagggttaa gtggggggtc tttaagattaaattctctga attgtacata catggttaca 1440 cggatattgt attcctggtc gtatatactgttttcgaacg cagtgccgag gcctacgtgg 1500 tctacatttc cagtagtttg tagtctcagccacagctgat ttcttttgtt gtttggttgg 1560 aagtaatcaa tagtggaatc taggacaggtttgggggtaa agtagcggga gtggtaggag 1620 aagggctggg ttatggtatg gcgggaggagtagtttacat aggggtcata ggtgagggct 1680 gtggcctttg ttacaaagtt atcatctagaataacagcac tggagcccac tcccctgtca 1740 ccctgggtga tcggggagca gggccag 17673 1768 DNA Porcine circovirus 3 aattcaacct taaccttttt tattctgtagtattcaaagg gtatagagat tttgttggtc 60 ccccctcccg ggggaacaaa gtcgtcaatattaaatctca tcatgtccac cgcccaggag 120 ggcgttctga ctgtggtagc cttgacagtatatccgaagg tgcgggagag gcgggtgttg 180 aagatgccat ttttccttct ccaacggtagcggtggcggg ggtggacgag ccaggggcgg 240 cggcggagga tctggccaag atggctgcgggggcggtgtc ttcttctgcg gtaacgcctc 300 cttggatacg tcatagctga aaacgaaagaagtgcgctgt aagtattacc agcgcacttc 360 ggcagcggca gcacctcggc agcacctcagcagcaacatg cccagcaaga agaatggaag 420 aagcggaccc caaccacata aaaggtgggtgttcacgctg aataatcctt ccgaagacga 480 gcgcaagaaa atacgggagc tcccaatctccctatttgat tattttattg ttggcgagga 540 gggtaatgag gaaggacgaa cacctcacctccaggggttc gctaattttg tgaagaagca 600 aacttttaat aaagtgaagt ggtatttgggtgcccgctgc cacatcgaga aagccaaagg 660 aactgatcag cagaataaag aatattgcagtaaagaaggc aacttactta ttgaatgtgg 720 agctcctcga tctcaaggac aacggagtgacctgtctact gctgtgagta ccttgttgga 780 gagcgggagt ctggtgaccg ttgcagagcagcaccctgta acgtttgtca gaaatttccg 840 cgggctggct gaacttttga aagtgagcgggaaaatgcag aagcgtgatt ggaagaccaa 900 tgtacacgtc attgtggggc cacctgggtgtggtaaaagc aaatgggctg ctaattttgc 960 agacccggaa accacatact ggaaaccacctagaaacaag tggtgggatg gttaccatgg 1020 tgaagaagtg gttgttattg atgacttttatggctggctg ccgtgggatg atctactgag 1080 actgtgtgat cgatatccat tgactgtagagactaaaggt ggaactgtac cttttttggc 1140 ccgcagtatt ctgattacca gcaatcagaccccgttggaa tggtactcct caactgctgt 1200 cccagctgta gaagctctct atcggaggattacttccttg gtattttgga agaatgctac 1260 agaacaatcc acggaggaag ggggccagttcgtcaccctt tcccccccat gccctgaatt 1320 tccatatgaa ataaattact gagtcttttttatcacttcg taatggtttt tattattcat 1380 ttagggttta agtggggggt ctttaagattaaattctctg aattgtacat acatggttac 1440 acggatattg tagtcctggt cgtatatactgttttcgaac gcagtgccga ggcctacgtg 1500 gtccacattt ctagaggttt gtagcctcagccaaagctga ttccttttgt tatttggttg 1560 gaagtaatca atagtggagt caagaacaggtttgggtgtg aagtaacggg agtggtagga 1620 gaagggttgg gggattgtat ggcgggaggagtagtttaca tatgggtcat aggttagggc 1680 tgtggccttt gttacaaagt tatcatctagaataacagca gtggagccca ctcccctatc 1740 accctgggtg atgggggagc agggccag1768 4 1768 DNA Porcine circovirus 4 aattcaacct taacctttct tattctgtagtattcaaagg gtatagagat tttgttggtc 60 ccccctcccg ggggaacaaa gtcgtcaattttaaatctca tcatgtccac cgcccaggag 120 ggcgttgtga ctgtggtacg cttgacagtatatccgaagg tgcgggagag gcgggtgttg 180 aagatgccat ttttccttct ccaacggtagcggtggcggg ggtggacgag ccaggggcgg 240 cggcggagga tctggccaag atggctgcgggggcggtgtc ttcttctgcg gtaacgcctc 300 cttggatacg tcatagctga aaacgaaagaagtgcgctgt aagtattacc agcgcacttc 360 ggcagcggca gcacctcggc agcacctcagcagcaacatg cccagcaaga agaatggaag 420 aagcggaccc caaccacata aaaggtgggtgttcacgctg aataatcctt ccgaagacga 480 gcgcaagaaa atacgggagc tcccaatctccctatttgat tattttattg ttggcgagga 540 gggtaatgag gaaggacgaa cacctcacctccaggggttc gctaattttg tgaagaagca 600 aacttttaat aaagtgaagt ggtatttgggtgcccgctgc cacatcgaga aagccaaagg 660 aactgatcag cagaataaag aatattgcagtaaagaaggc aacttactta ttgaatgtgg 720 agctcctcga tctcaaggac aacggagtgacctgtctact gctgtgagta ccttgttgga 780 gagcgggagt ctggtgaccg ttgcagagcagcaccctgta acgtttgtca gaaatttccg 840 cgggctggct gaacttttga aagtgagcgggaaaatgcag aagcgtgatt ggaagaccaa 900 tgtacacgtc attgtggggc cacctgggtgtggtaaaagc aaatgggctg ctaattttgc 960 agacccggaa accacatact ggaaaccacctagaaacaag tggtgggatg gttaccatgg 1020 tgaagaagtg gttgttattg atgacttttatggctggctg ccgtgggatg atctactgag 1080 actgtgtgat cgatatccat tgactgtagagactaaaggt ggaactgtac cttttttggc 1140 ccgcagtatt ctgattacca gcaatcagaccccgttggaa tggtactcct caactgctgt 1200 cccagctgta gaagctctct atcggaggattacttccttg gtattttgga agaatgctac 1260 agaacaatcc acggaggaag ggggccagttcgtcaccctt tcccccccat gccctgaatt 1320 tccatatgaa ataaattact gagtcttttttatcacttcg taatggtttt tattattcat 1380 ttagggttta agtggggggt ctttaagattaaattctctg aattgtacat acatggttac 1440 acggatattg tagtcctggt cgtatttactgttttcgaac gcagcgccga ggcctacgtg 1500 gtccacattt ccagaggttt gtagtctcagccaaagctga ttccttttgt tatttggttg 1560 gaagtaatca atagtggagt caagaacaggtttgggtgtg aagtaacggg agtggtagga 1620 gaagggttgg gggattgtat ggcgggaggagtagtttaca tatgggtcat aggttagggc 1680 tgtggccttt gttacaaagt tatcatctagaataacagca gtggagccca ctcccctatc 1740 accctgggtg atgggggagc agggccag1768 5 1759 DNA Porcine circovirus 5 aattcatatt tagcctttct aatacggtagtattggaaag gtaggggtag ggggttggtg 60 ccgcctgagg gggggaggaa ctggccgatgttgaatttga ggtagttaac attccaagat 120 ggctgcgagt atcctccttt tatggtgagtacaaattctg tagaaaggcg ggaattgaag 180 atacccgtct ttcggcgcca tctgtaacggtttctgaagg cggggtgtgc caaatatggt 240 cttctccgga ggatgtttcc aagatggctgcgggggcggg tccttcttct gcggtaacgc 300 ctccttggcc acgtcatcct ataaaagtgaaagaagtgcg ctgctgtagt attaccagcg 360 cacttcggca gcggcagcac ctcggcagcgtcagtgaaaa tgccaagcaa gaaaagcggc 420 ccgcaacccc ataagaggtg ggtgttcacccttaataatc cttccgagga ggagaaaaac 480 aaaatacggg agcttccaat ctccctttttgattattttg tttgcggaga ggaaggtttg 540 gaagagggta gaactcctca cctccaggggtttgcgaatt ttgctaagaa gcagactttt 600 aacaaggtga agtggtattt tggtgcccgctgccacatcg agaaagcgaa aggaaccgac 660 cagcagaata aagaatactg cagtaaagaaggccacatac ttatcgagtg tggagctccg 720 cggaaccagg ggaagcgcag cgacctgtctactgctgtga gtaccctttt ggagacgggg 780 tctttggtga ctgtagccga gcagttccctgtaacgtatg tgagaaattt ccgcgggctg 840 gctgaacttt tgaaagtgag cgggaagatgcagcagcgtg attggaagac agctgtacac 900 gtcatagtgg gcccgcccgg ttgtgggaagagccagtggg cccgtaattt tgctgagcct 960 agggacacct actggaagcc tagtagaaataagtggtggg atggatatca tggagaagaa 1020 gttgttgttt tggatgattt ttatggctggttaccttggg atgatctact gagactgtgt 1080 gaccggtatc cattgactgt agagactaaagggggtactg ttcctttttt ggcccgcagt 1140 attttgatta ccagcaatca ggccccccaggaatggtact cctcaactgc tgtcccagct 1200 gtagaagctc tctatcggag gattactactttgcaatttt ggaagactgc tggagaacaa 1260 tccacggagg tacccgaagg ccgatttgaagcagtggacc caccctgtgc ccttttccca 1320 tataaaataa attactgagt cttttttgttatcacatcgt aatggttttt atttttattt 1380 atttagaggg tcttttagga taaattctctgaattgtaca taaatagtca gccttaccac 1440 ataattttgg gctgtggctg cattttggagcgcatagccg aggcctgtgt gctcgacatt 1500 ggtgtgggta tttaaatgga gccacagctggtttctttta ttatttgggt ggaaccaatc 1560 aattgtttgg tccagctcag gtttgggggtgaagtacctg gagtggtagg taaagggctg 1620 ccttatggtg tggcgggagg agtagttaatataggggtca taggccaagt tggtggaggg 1680 ggttacaaag ttggcatcca agataacaacagtggaccca acacctcttt gattagaggt 1740 gatggggtct ctggggtaa 1759 6 1768DNA Porcine circovirus misc_feature (1)..(1768) nucleotide “n” can beeither of the nucleotides ′a“, ”c“, ”g′ or “t” 6 gaattcaacc ttaaccttttttattctgta gtattcaaag ggtataaaga ttttgttggt 60 cccccctccc gggggaacaaagtcgtcaat attaaatctc atcatgtcca ccgcccagga 120 gggcgttctg actgtggtagccttgacagt atatccgaag gtgcgggaga rgcgggtgtt 180 gaaaatgcca tttttccttctccaacggta gcggtggcgg gggtggacma nccacgggcg 240 gcggcggawg atctggccaagatggctgcg ggggcggtgt cttcttctgc ggtaacgcct 300 ccttggatac gtcatagctgaaaacgaaag aagtgcgctg taagtattac cagcgcactt 360 cggcagcggc agcacctcggcagcacctca gcagcaacat gcccagcaag aagaatggaa 420 gaagcggacc ccaaccacataaaaggtggg tgttcacgct gaataatcct tccgaagacg 480 agcgcaagaa aatacgggagctcccaatct ccctatttga ttattttatt gttggcgagg 540 agggtwwtga ggaangacgaacacctcacc tccaggggtt cgctaatttt gtgaagaagc 600 aaacttttaa taaagtgaagtggtatttgg gtgcccgctg ccacatcgag aaagccaaag 660 gaactgatca gcagaataaagaatattgca gtaaagaagg caacttactt attgaatgtg 720 gagctcctcg atctcaaggacaacggagtg acctgtctac tgctgtgagt accttgttgg 780 agagcgggag tctggtgaccgttgcagagc agcaccctgt aacgtttgtc agaaatttcc 840 gcgggctggc tgaacttttgaaagtgagcg ggaaaatgca gaagcgtgat tggaagacca 900 atgtacacgt cattgtggggccacctgggt gtggtaaaag caaatgggct gctaattttg 960 cagacccgga aaccacatactggaaaccac ctagaaacaa gtggtgggat ggttaccatg 1020 gtgaagaagt ggttgttattgatgactttt atggctggct gccgtgggat gatctactga 1080 gactgtgtga tcgatatccattgactgtag agactaaagg tggaactgta cnnnnnnngg 1140 cccgcagtat tctgattaccagcaatcaga ccccgttgga atggtactcc tcaactgctg 1200 tcccagctgt agaagctctctatcggagga ttacttcctt ggtattttgg aagaatgcta 1260 cagaacaatc cacggaggaagggggccagt tngtcaccct ttccccccca tgccctgaat 1320 ttccatatga aataaattactgagtctttt ttatcacttc gtaatggttt ttattattca 1380 tttagggttt aagtggggggtctttaagat taaattctct gaattgtaca tacatggtta 1440 cacggatatt gtagtcctggtcgtatatac tgttttcgaa cgcagtgccg aggcctacgt 1500 ggtccacatt tctagaggtttgtagcctca gccaaagctg attccttttg ttatttggtt 1560 ggaagtaatc aatagtggagtcaagaacag gtttgggtgt gaagtaacgg gagtggtagg 1620 agaagggttg ggggattgtatggcgggagg agtagtttac atatgggtca taggttaggg 1680 ctgtggcctt tgttacaaagttatcatcta gaataacagc agtggagccc actcccctat 1740 caccctgggt gatgggggagcagggcca 1768 7 1768 DNA Porcine circovirus misc_feature (1)..(1768)nucleotide “k” can be either “g” or “t” and nucleotide “y” can be either“c” or “t” 7 accagcgcac ttcggcagcg gcagcacctc ggcagcacct cagcagcaacatgcccagca 60 agaagaatgg aagaagcgga ccccaaccac ataaaaggtg ggtgttcacgctgaataatc 120 cttccgaaga cgagcgcaag aaaatacggg agctcccaat ctccctatttgattatttta 180 ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccaggggttcgctaatt 240 ttgtgaagaa kcaaactttt aataaagtga agtggtattt gggtgcccgctgccacatcg 300 agaaagccaa aggaactgat cagcagaata aagaatattg cagtaaagaaggcaacttac 360 ttattgaatg tggagctcct cgatctcaag gacaacggag tgacctgtctactgctgtga 420 gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccctgtaacgtttg 480 tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatgcagaagcgtg 540 attggaagac caatgtacac gtcattgtgg ggccacctgg gtgtggtaaaagcaaatggg 600 ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaacaagtggtggg 660 atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctggctgccgtggg 720 atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaaggtggaactg 780 tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttggaatggtact 840 cctcaactgc tgtcccagct gtagaagctc tctatcggag gattacttccttggtatttt 900 ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcaccctttcccccc 960 catgccctga atttccatat gaaataaatt actgagtcyt ttttatcacttcgtaatggt 1020 ttttattatt catttagggg ttaagtgggg ggtctttaag attaaattccctgaattgta 1080 catacagggt tacacggata ttgtagtcct ggtcgtattt actgttttcgaacgcagtgc 1140 cgaggcctac gtggtccaca tttctagagg tttgtagcct cagccaaagctgattccttt 1200 tgttatttgg ttggaagtaa tcaatagtgg agtcaagaac aggtttgggtgtgaagtaac 1260 gggagtggta ggagaagggt tgggggattg tatggcggga ggagtagtttacatatgggt 1320 catatgtttg ggctgtggcc tttggtacaa agttatcatc tagaataacagcagtggagc 1380 ccactcccct atcaccctgg gtgatggggg agcagggcca gaattcaaccttaacctttc 1440 ttattctgta gtattcaaag ggtatagaga ttttgttggt cccccctcccgggggaacaa 1500 agtcgtcaat tttaaatctc atcatgtcca ccgcccagga gggcgttgtgactgtggtac 1560 gcttgacagt atatccgaag gtgcgggaga ggcgggtgtt gaagatgccatttttccttc 1620 tccaacggta gcggtggcgg gggtggacga gccaggggcg gcggcggaggatctggccaa 1680 gatggctgcg ggggcggtgt cttcttctgc ggtaacgcct ccttggatatgtcatagctg 1740 aaaacgaaag aagtgcgctg taagtatt 1768 8 1768 DNA Porcinecircovirus 8 accagcgcac ttcggcagcg gcagcacctc ggcagcacct cagcagcaacatgcccagca 60 agaagaatgg aagaagcgga ccccaaccac ataaaaggtg ggtgttcacgctgaataatc 120 cttccgaaga cgagcgcaag aaaatacggg agctcccaat ctccctatttgattatttta 180 ttgttggcga ggagggtaat gaggaaggac gaacacctca cctccaggggttcgctaatt 240 ttgtgaagaa gcaaactttt aataaagtga agtggtattt gggtgcccgctgccacatcg 300 agaaagccaa aggaactgat cagcagaata aagaatattg cagtaaagaaggcaacttac 360 ttattgaatg tggagctcct cgatctcaag gacaacggag tgacctgtctactgctgtga 420 gtaccttgtt ggagagcggg agtctggtga ccgttgcaga gcagcaccctgtaacgtttg 480 tcagaaattt ccgcgggctg gctgaacttt tgaaagtgag cgggaaaatgcagaagcgtg 540 attggaagac caatgtacac gtcattgtgg ggccacctgg gtgtggtaaaagcaaatggg 600 ctgctaattt tgcagacccg gaaaccacat actggaaacc acctagaaacaagtggtggg 660 atggttacca tggtgaagaa gtggttgtta ttgatgactt ttatggctggctgccgtggg 720 atgatctact gagactgtgt gatcgatatc cattgactgt agagactaaaggtggaactg 780 tacctttttt ggcccgcagt attctgatta ccagcaatca gaccccgttggaatggtact 840 cctcaactgc tgtcccagct gtagaagctc tctatcggag gattacttccttggtatttt 900 ggaagaatgc tacagaacaa tccacggagg aagggggcca gttcgtcaccctttcccccc 960 catgccctga atttccatat gaaataaatt actgagtctt ttttatcacttcgtaatggt 1020 ttttattatt catttagggg ttaagtgggg ggtctttaag attaaattctctgaattgta 1080 catacatggt tacacggata ttgtagtcct ggtcgtattt actgttttcgaacgcagtgc 1140 cgaggcctac gtggtccaca tttctagagg tttgtagcct cagccaaagctgattccttt 1200 tgttatttgg ttggaagtaa tcaatagtgg agtcaagaac aggtttgggtgtgaagtaac 1260 gggagtggta ggagaagggt tgggggattg tatggcggga ggagtagtttacatatgggt 1320 cataggttag ggctgtggcc tttggtacaa agttatcatc tagaataacagcagtggagc 1380 ccactcccct atcaccctgg gtgatggggg agcagggcca gaattcaaccttaacctttt 1440 ttattctgta gtattcaaag ggtatagaga ttttgttggt cccccctcccgggggaacaa 1500 agtcgtcaat tttaaatctc atcatgtcca ccgcccagga gggcgttgtgactgtagtac 1560 gcttgacagt atatccgaag gtgcgggaga ggcgggtgtt gaagatgccatttttccttc 1620 tccaacggta gcggtggcgg gggtggacga gccaggggcg gcggcggaggatctggccaa 1680 gatggctgcg ggggcggtgt cttcttctgc ggtaacgcct ccttggatacgtcatagctg 1740 aaaacgaaag aagtgcgctg taagtatt 1768

What is claimed is:
 1. A composition for reducing viral load of porcinecircovirus-2 (PCV-2) comprising a pharmaceutically or veterinarily ormedically acceptable carrier and an active agent comprising a PCV-2immunogen present in an amount effective to reduce viral load, whereinsaid immunogen is an inactivated PCV-2.
 2. The composition of claim 1,wherein the immunogen is recombinantly produced.
 3. A method forreducing viral load of porcine circovirus-2 (PCV-2) in a pig comprisingadministering to the pig the composition of claim
 1. 4. The method ofclaim 3, wherein the immunogen is recombinantly produced.
 5. The methodof claim 3, wherein the administering is prior to breeding.
 6. Themethod of claim 3, wherein the administering is to a pregnant femalepig.